Insights from transcriptome profiling on the non-photosynthetic and stomatal signaling response of maize carbonic anhydrase mutants to low CO2.

BACKGROUND: Carbonic anhydrase (CA) catalyzes the hydration of CO2 in the first biochemical step of C4 photosynthesis, and has been considered a potentially rate-limiting step when CO2 availability within a leaf is low. Previous work in Zea mays (maize) with a double knockout of the two highest-expressed ß-CA genes, CA1 and CA2, reduced total leaf CA activity to less than 3% of wild-type. Surprisingly, this did not limit photosynthesis in maize at ambient or higher CO2concentrations. However, the ca1ca2 mutants exhibited reduced rates of photosynthesis at sub-ambient CO2, and accumulated less biomass when grown under sub-ambient CO2 (9.2 Pa). To further clarify the importance of CA for C4 photosynthesis, we assessed gene expression changes in wild-type, ca1 and ca1ca2 mutants in response to changes in pCO2 from 920 to 9.2 Pa. RESULTS: Leaf samples from each genotype were collected for RNA-seq analysis at high CO2 and at two time points after the low CO2 transition, in order to identify early and longer-term responses to CO2 deprivation. Despite the existence of multiple isoforms of CA, no other CA genes were upregulated in CA mutants. Although photosynthetic genes were downregulated in response to low CO2, differential expression was not observed between genotypes. However, multiple indicators of carbon starvation were present in the mutants, including amino acid synthesis, carbohydrate metabolism, and sugar signaling. In particular, multiple genes previously implicated in low carbon stress such as asparagine synthetase, amino acid transporters, trehalose-6-phosphate synthase, as well as many transcription factors, were strongly upregulated. Furthermore, genes in the CO2 stomatal signaling pathway were differentially expressed in the CA mutants under low CO2. CONCLUSIONS: Using a transcriptomic approach, we showed that carbonic anhydrase mutants do not compensate for the lack of CA activity by upregulating other CA or photosynthetic genes, but rather experienced extreme carbon stress when grown under low CO2. Our results also support a role for CA in the CO2 stomatal signaling pathway. This study provides insight into the importance of CA for C4 photosynthesis and its role in stomatal signaling.

Carbonic Anhydrase Mutants in Zea mays Have Altered Stomatal Responses to Environmental Signals.

Stomata regulate transpirational water loss and CO2 uptake for photosynthesis in response to changing environmental conditions. Research investigating stomatal movement has mostly been conducted in C3 eudicot species, which have very different CO2 requirements for photosynthesis relative to C4 grasses. Carbonic anhydrase (CA) catalyzes the hydration of CO2, and its activity has been linked to stomatal aperture regulation in eudicots. The number of Ca genes and their evolutionary history differ between monocots and dicots, and many questions remain unanswered about potential neofunctionalization and subfunctionalization of grass Ca paralogs and their roles in photosynthesis and stomatal conductance. To investigate the roles of different Ca genes in maize (Zea mays), we examined stomatal responses in ca1 and ca2 single mutants as well as a ca1ca2 double mutant. The ca1 and ca2 single mutants had 10% and 87% of the CA activity exhibited by the wild type, respectively, while ca1ca2 had less than 5% of wild-type CA activity. The ca mutants had higher stomatal conductance than the wild type and slower stomatal closure in response to increases in CO2 partial pressure. Contrary to previous reports in eudicots, ca mutants showed slowed stomatal closure in response to the light-dark transition and did not show differences in stomatal density compared with the wild type. These results implicate CA-mediated signaling in the control of stomatal movement but not stomatal development. Drought experiments with ca1ca2 mutant plants suggest a role for CA in water-use efficiency and reveal that Z. mays is not optimized for water-use efficiency under well-watered conditions.

Mesophyll conductance in Zea mays responds transiently to CO2 availability: implications for transpiration efficiency in C4 crops.

Mesophyll conductance (gm ) describes the movement of CO2 from the intercellular air spaces below the stomata to the site of initial carboxylation in the mesophyll. In contrast with C3 -gm , little is currently known about the intraspecific variation in C4 -gm or its responsiveness to environmental stimuli. To address these questions, gm was measured on five maize (Zea mays) lines in response to CO2 , employing three different estimates of gm . Each of the methods indicated a significant response of gm to CO2 . Estimates of gm were similar between methods at ambient and higher CO2 , but diverged significantly at low partial pressures of CO2 . These differences are probably driven by incomplete chemical and isotopic equilibrium between CO2 and bicarbonate under these conditions. Carbonic anhydrase and phosphoenolpyruvate carboxylase in vitro activity varied significantly despite similar values of gm and leaf anatomical traits. These results provide strong support for a CO2 response of gm in Z. mays, and indicate that gm in maize is probably driven by anatomical constraints rather than by biochemical limitations. The CO2 response of gm indicates a potential role for facilitated diffusion in C4 -gm . These results also suggest that water-use efficiency could be enhanced in C4 species by targeting gm .

A Limited Role for Carbonic Anhydrase in C4 Photosynthesis as Revealed by a ca1ca2 Double Mutant in Maize.

Carbonic anhydrase (CA) catalyzes the first biochemical step of the carbon-concentrating mechanism of C4 plants, and in C4 monocots it has been suggested that CA activity is near limiting for photosynthesis. Here, we test this hypothesis through the characterization of transposon-induced mutant alleles of Ca1 and Ca2 in maize (Zea mays). These two isoforms account for more than 85% of the CA transcript pool. A significant change in isotopic discrimination is observed in mutant plants, which have as little as 3% of wild-type CA activity, but surprisingly, photosynthesis is not reduced under current or elevated CO2 partial pressure (pCO2). However, growth and rates of photosynthesis under subambient pCO2 are significantly impaired in the mutants. These findings suggest that, while CA is not limiting for C4 photosynthesis in maize at current pCO2, it likely maintains high rates of photosynthesis when CO2 availability is reduced. Current atmospheric CO2 levels now exceed 400 ppm (approximately 40.53 Pa) and contrast with the low-pCO2 conditions under which C4 plants expanded their range approximately 10 million years ago, when the global atmospheric CO2 was below 300 ppm (approximately 30.4 Pa). Thus, as CO2 levels continue to rise, selective pressures for high levels of CA may be limited to arid climates where stomatal closure reduces CO2 availability to the leaf.

Comparative Analysis of Metagenomics and Metataxonomics for the Characterization of Vermicompost Microbiomes.

The study of microbial communities or microbiotas in animals and environments is important because of their impact in a broad range of industrial applications, diseases and ecological roles. High throughput sequencing (HTS) is the best strategy to characterize microbial composition and function. Microbial profiles can be obtained either by shotgun sequencing of genomes, or through amplicon sequencing of target genes (e.g., 16S rRNA for bacteria and ITS for fungi). Here, we compared both HTS approaches at assessing taxonomic and functional diversity of bacterial and fungal communities during vermicomposting of white grape marc. We applied specific HTS workflows to the same 12 microcosms, with and without earthworms, sampled at two distinct phases of the vermicomposting process occurring at 21 and 63 days. Metataxonomic profiles were inferred in DADA2, with bacterial metabolic pathways predicted via PICRUSt2. Metagenomic taxonomic profiles were inferred in PathoScope, while bacterial functional profiles were inferred in Humann2. Microbial profiles inferred by metagenomics and metataxonomics showed similarities and differences in composition, structure, and metabolic function at different taxonomic levels. Microbial composition and abundance estimated by both HTS approaches agreed reasonably well at the phylum level, but larger discrepancies were observed at lower taxonomic ranks. Shotgun HTS identified ~1.8 times more bacterial genera than 16S rRNA HTS, while ITS HTS identified two times more fungal genera than shotgun HTS. This is mainly a consequence of the difference in resolution and reference richness between amplicon and genome sequencing approaches and databases, respectively. Our study also revealed great differences and even opposite trends in alpha- and beta-diversity between amplicon and shotgun HTS. Interestingly, amplicon PICRUSt2-imputed functional repertoires overlapped ~50% with shotgun Humann2 profiles. Finally, both approaches indicated that although bacteria and fungi are the main drivers of biochemical decomposition, earthworms also play a key role in plant vermicomposting. In summary, our study highlights the strengths and weaknesses of metagenomics and metataxonomics and provides new insights on the vermicomposting of white grape marc. Since both approaches may target different biological aspects of the communities, combining them will provide a better understanding of the microbiotas under study.

Physician Understanding and Willingness to Prescribe Biosimilars: Findings from a US National Survey.

BACKGROUND: Biosimilars have the potential to increase patient access and significantly reduce healthcare costs in the US. However, uptake in the US has been slower than anticipated, limiting the benefits of biosimilar competition. Understanding the factors that affect uptake is critically important to realize the benefits of biosimilars. METHODS: A US national survey study was conducted electronically from December 11, 2019 to January 8, 2020. The survey was administered to 507 US healthcare professionals practicing in dermatology, gastroenterology, hematology, oncology, nephrology, or rheumatology. The survey evaluated prescriber attitudes toward biosimilars in general, as well as prescriber decision making, using a series of hypothetical scenarios with fictional biological products. RESULTS: Fewer than half had a baseline understanding of key elements of biosimilarity, even among respondents who had previously prescribed a biosimilar. Regardless of previous experience, all respondents benefited from receiving additional information about biosimilarity, indicating the potential benefits of educational efforts for prescribers across all specialties and levels of experience. Prescriber choice was driven primarily by formulary status; however, respondents identified a variety of factors that would influence their willingness to prescribe a biosimilar, including financial savings to the patient, pharmacovigilance, patient experience, and education on the FDA approval process. Over one-third of participants indicated a preference for reference products and nearly half indicated a hesitancy to try biosimilars until they have been on the market longer. Naming conventions for biosimilars did not affect prescribers' willingness to prescribe biosimilars. CONCLUSIONS: Gaps in prescriber knowledge and hesitancy toward biosimilars remain significant challenges for biosimilar uptake. While formulary status of a biosimilar product strongly influences prescriber choice, additional prescriber education on biosimilarity is needed.

Comparative analysis of the bronchoalveolar microbiome in Portuguese patients with different chronic lung disorders.

The lung is inhabited by a diverse microbiome that originates from the oropharynx by a mechanism of micro-aspiration. Its bacterial biomass is usually low; however, this condition shifts in lung cancer (LC), chronic obstructive pulmonary disease (COPD) and interstitial lung disease (ILD). These chronic lung disorders (CLD) may coexist in the same patient as comorbidities and share common risk factors, among which the microbiome is included. We characterized the microbiome of 106 bronchoalveolar lavages. Samples were initially subdivided into cancer and non-cancer and high-throughput sequenced for the 16S rRNA gene. Additionally, we used a cohort of 25 CLD patients where crossed comorbidities were excluded. Firmicutes, Proteobacteria and Bacteroidetes were the most prevalent phyla independently of the analyzed group. Streptococcus and Prevotella were associated with LC and Haemophilus was enhanced in COPD versus ILD. Although no significant discrepancies in microbial diversity were observed between cancer and non-cancer samples, statistical tests suggested a gradient across CLD where COPD and ILD displayed the highest and lowest alpha diversities, respectively. Moreover, COPD and ILD were separated in two clusters by the unweighted UniFrac distance (P value = 0.0068). Our results support the association of Streptoccocus and Prevotella with LC and of Haemophilus with COPD, and advocate for specific CLD signatures.

Human Endogenous Retrovirus Expression Is Associated with Head and Neck Cancer and Differential Survival.

Human endogenous retroviruses (HERVs) have been implicated in a variety of human diseases including cancers. However, technical challenges in analyzing HERV sequence data have limited locus-specific characterization of HERV expression. Here, we use the software Telescope (developed to identify expressed transposable elements from metatranscriptomic data) on 43 paired tumor and adjacent normal tissue samples from The Cancer Genome Atlas Program to produce the first locus-specific retrotranscriptome of head and neck cancer. Telescope identified over 3000 expressed HERVs in tumor and adjacent normal tissue, and 1078 HERVs were differentially expressed between the two tissue types. The majority of differentially expressed HERVs were expressed at a higher level in tumor tissue. Differentially expressed HERVs were enriched in members of the HERVH family. Hierarchical clustering based on HERV expression in tumor-adjacent normal tissue resulted in two distinct clusters with significantly different survival probability. Together, these results highlight the importance of future work on the role of HERVs across a range of cancers.

Altered Middle Ear Microbiome in Children With Chronic Otitis Media With Effusion and Respiratory Illnesses.

Chronic otitis media with effusion (COME) is a common childhood disease characterized by an accumulation of fluid behind the eardrum. COME often requires surgical intervention and can also lead to significant hearing loss and subsequent learning disabilities. Recent characterization of the middle ear fluid (MEF) microbiome in pediatric patients has led to an improved understanding of the microbiota present in the middle ear during COME. However, it is not currently known how the MEF microbiome might vary due to other conditions, particularly respiratory disorders. Here, we apply an amplicon sequence variant (ASV) pipeline to MEF 16S rRNA high-throughput sequencing data from 50 children with COME (ages 3-176 months) undergoing tube placement. We achieve a more detailed taxonomic resolution than previously reported, including species and genus level resolution. Additionally, we provide the first report of the functional roles of the MEF microbiome and demonstrate that despite high taxonomic diversity, the functional capacity of the MEF microbiome remains uniform between patients. Furthermore, we analyze microbiome differences between children with COME with and without a history of lower airway disease (i.e., asthma or bronchiolitis). The MEF microbiome was less diverse in participants with lower airway disease than in patients without, and phylogenetic ß-diversity (weighted UniFrac) was significantly different based on lower airway disease status. Differential abundance between patients with lower airway disease and those without was observed for the genera Haemophilus, Moraxella, Staphylococcus, Alloiococcus, and Turicella. These findings support previous suggestions of a link between COME and respiratory illnesses and emphasize the need for future study of the middle ear and respiratory tract microbiomes in diseases such as asthma and bronchiolitis.

Changes in the composition and function of bacterial communities during vermicomposting may explain beneficial properties of vermicompost.

Vermicomposting is the process by which organic waste is broken down through the synergistic actions of earthworms and microbial communities. Although vermicomposting has been shown to effectively reduce organic biomass and generate high-quality fertilizer for plants, little is known about the bacterial communities that are involved in this decomposition process. Since optimization of vermicomposting for commercial use necessitates additional knowledge of the underlying biological processes, this study sought to characterize the bacterial succession involved in the vermicomposting of Scotch broom (Cytisus scoparius), a leguminous shrub that has become invasive around the world with consequences for the dynamics and productivity of the ecosystems they occupy. Scotch broom was processed in a pilot-scale vermireactor for 91 days with the earthworm species Eisenia andrei. Samples were taken at the initiation of vermicomposting, and days 14, 42 and 91, representing both active and mature stages of vermicomposting. Significant changes (P < 0.0001) in the bacterial community composition (richness and evenness) were observed throughout the process. Increases in taxonomic diversity were accompanied by increases in functional diversity of the bacterial community, including metabolic capacity, streptomycin and salicylic acid synthesis, and nitrification. These results highlight the role of bacterial succession during the vermicomposting process and provide evidence of microbial functions that may explain the beneficial effects of vermicompost on soil and plants.

Bacterial succession and functional diversity during vermicomposting of the white grape marc Vitis vinifera v. Albariño.

Winemaking produces millions of tons of grape marc, a byproduct of grape pressing, each year. Grape marc is made up of the skins, stalks, and seeds remaining after pressing. Raw grape marc can be hazardous to the environment due to its low pH and high polyphenol content, but previous work has shown that grape marc can be stabilized via vermicomposting to produce organic fertilizer. Here, we utilize 16S rRNA high-throughput sequencing to characterize the bacterial community composition, diversity and metabolic function during vermicomposting of the white grape marc Vitis vinifera v. Albariño for 91 days. Large, significant changes in the bacterial community composition of grape marc vermicompost were observed by day 7 of vermicomposting and throughout the duration of the experiment until day 91. Similarly, taxonomic and phylogenetic α-diversity increased throughout the experiment and estimates of ß-diversity differed significantly between time points. Functional diversity also changed during vermicomposting, including increases in cellulose metabolism, plant hormone synthesis, and antibiotic synthesis. Thus, vermicomposting of white grape marc resulted in a rich, stable bacterial community with functional properties that may aid plant growth. These results support the use of grape marc vermicompost for sustainable agricultural practices in the wine industry.

Rapid Bacterial Community Changes during Vermicomposting of Grape Marc Derived from Red Winemaking.

Previous studies dealing with changes in microbial communities during vermicomposting were mostly performed at lab-scale conditions and by using low-throughput techniques. Therefore, we sought to characterize the bacterial succession during the vermicomposting of grape marc over a period of 91 days in a pilot-scale vermireactor. Samples were taken at the initiation of vermicomposting, and days 14, 28, 42, and 91, representing both active and mature stages of vermicomposting. By using 16S rRNA high-throughput sequencing, significant changes in the bacterial community composition of grape marc were found after 14 days and throughout the process (p < 0.0001). There was also an increase in bacterial diversity, both taxonomic and phylogenetic, from day 14 until the end of the trial. We found the main core microbiome comprised of twelve bacterial taxa (~16.25% of the total sequences) known to be capable of nitrogen fixation and to confer plant-disease suppression. Accordingly, functional diversity included increases in specific genes related to nitrogen fixation and synthesis of plant hormones (salicylic acid) after 91 days. Together, the findings support the use of grape marc vermicompost for sustainable practices in the wine industry by disposing of this high-volume winery by-product and capturing its value to improve soil fertility.

Biochemical and transcriptomic analysis of maize diversity to elucidate drivers of leaf carbon isotope composition.

Carbon isotope discrimination is used to study CO2 diffusion, substrate availability for photosynthesis, and leaf biochemistry, but the intraspecific drivers of leaf carbon isotope composition (δ13C) in C4 species are not well understood. In this study, the role of photosynthetic enzymes and post-photosynthetic fractionation on δ13C (‰) was explored across diverse maize inbred lines. A significant 1.3‰ difference in δ13C was observed between lines but δ13C did not correlate with in vitro leaf carbonic anhydrase (CA), phosphoenolpyruvate carboxylase (PEPC), or ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activity. RNA-sequencing was used to identify potential differences in post-photosynthetic metabolism that would influence δ13C; however, no correlations were identified that would indicate significant differences in post-photosynthetic fractionation between lines. Variation in δ13C has been observed between C4 subtypes, but differential expression of NADP-ME and PEP-CK pathways within these lines did not correlate with δ13C. However, co-expression network analysis provided novel evidence for isoforms of C4 enzymes and putative transporters. Together, these data indicate that diversity in maize δ13C cannot be fully explained by variation in CA, PEPC, or Rubisco activity or gene expression. The findings further emphasise the need for future work exploring the influence of stomatal sensitivity and mesophyll conductance on δ13C in maize.

The draft genome of the C3 panicoid grass species Dichanthelium oligosanthes.

BACKGROUND: Comparisons between C3 and C4 grasses often utilize C3 species from the subfamilies Ehrhartoideae or Pooideae and C4 species from the subfamily Panicoideae, two clades that diverged over 50 million years ago. The divergence of the C3 panicoid grass Dichanthelium oligosanthes from the independent C4 lineages represented by Setaria viridis and Sorghum bicolor occurred approximately 15 million years ago, which is significantly more recent than members of the Bambusoideae, Ehrhartoideae, and Pooideae subfamilies. D. oligosanthes is ideally placed within the panicoid clade for comparative studies of C3 and C4 grasses. RESULTS: We report the assembly of the nuclear and chloroplast genomes of D. oligosanthes, from high-throughput short read sequencing data and a comparative transcriptomics analysis of the developing leaf of D. oligosanthes, S. viridis, and S. bicolor. Physiological and anatomical characterizations verified that D. oligosanthes utilizes the C3 pathway for carbon fixation and lacks Kranz anatomy. Expression profiles of transcription factors along developing leaves of D. oligosanthes and S. viridis were compared with previously published data from S. bicolor, Zea mays, and Oryza sativa to identify a small suite of transcription factors that likely acquired functions specifically related to C4 photosynthesis. CONCLUSIONS: The phylogenetic location of D. oligosanthes makes it an ideal C3 plant for comparative analysis of C4 evolution in the panicoid grasses. This genome will not only provide a better C3 species for comparisons with C4 panicoid grasses, but also highlights the power of using high-throughput sequencing to address questions in evolutionary biology.