Absence of carbonic anhydrase in chloroplasts affects C3 plant development but not photosynthesis.

The enzyme carbonic anhydrase (CA), which catalyzes the interconversion of bicarbonate with carbon dioxide (CO2) and water, has been hypothesized to play a role in C3 photosynthesis. We identified two tobacco stromal CAs, ß-CA1 and ß-CA5, and produced CRISPR/Cas9 mutants affecting their encoding genes. While single knockout lines Δß-ca1 and Δß-ca5 had no striking phenotypic differences compared to wild type (WT) plants, Δß-ca1ca5 leaves developed abnormally and exhibited large necrotic lesions even when supplied with sucrose. Leaf development of Δß-ca1ca5 plants normalized at 9,000 ppm CO2 Leaves of Δß-ca1ca5 mutants and WT that had matured in high CO2 had identical CO2 fixation rates and photosystem II efficiency. Fatty acids, which are formed through reactions with bicarbonate substrates, exhibited abnormal profiles in the chloroplast CA-less mutant. Emerging Δß-ca1ca5 leaves produce reactive oxygen species in chloroplasts, perhaps due to lower nonphotochemical quenching efficiency compared to WT. Δß-ca1ca5 seedling germination and development is negatively affected at ambient CO2 Transgenes expressing full-length ß-CA1 and ß-CA5 proteins complemented the Δß-ca1ca5 mutation but inactivated (ΔZn-ßCA1) and cytoplasm-localized (Δ62-ßCA1) forms of ß-CA1 did not reverse the growth phenotype. Nevertheless, expression of the inactivated ΔZn-ßCA1 protein was able to restore the hypersensitive response to tobacco mosaic virus, while Δß-ca1 and Δß-ca1ca5 plants failed to show a hypersensitive response. We conclude that stromal CA plays a role in plant development, likely through providing bicarbonate for biosynthetic reactions, but stromal CA is not needed for maximal rates of photosynthesis in the C3 plant tobacco.

Student Performance along Axes of Scenario Novelty and Complexity in Introductory Biology: Lessons from a Unique Factorial Approach to Assessment.

In an undergraduate introductory biology laboratory course, we used a summative assessment to directly test the learning objective that students will be able to apply course material to increasingly novel and complex situations. Using a factorial framework, we developed multiple true-false questions to fall along axes of novelty and complexity, which resulted in four categories of questions: familiar content and low complexity (category A); novel content and low complexity (category B); familiar content and high complexity (category C); and novel content and high complexity (category D). On average, students scored more than 70% on all questions, indicating that the course largely met this learning objective. However, students scored highest on questions in category A, likely because they were most similar to course content, and lowest on questions in categories C and D. While we anticipated students would score equally on questions for which either novelty or complexity was altered (but not both), we observed that student scores in category C were lower than in category B. Furthermore, students performed equally poorly on all questions for which complexity was higher (categories C and D), even those containing familiar content, suggesting that application of course material to increasingly complex situations is particularly challenging to students.

A comparative transcriptomic study of an allotetraploid and its diploid progenitors illustrates the unique advantages and challenges of RNA-seq in plant species.

PREMISE OF THE STUDY: RNA-seq analysis of plant transcriptomes poses unique challenges due to the highly duplicated nature of plant genomes. We address these challenges in the context of recently formed polyploid species and detail an RNA-seq experiment comparing the leaf transcriptome profile of an allopolyploid relative of soybean with the diploid species that contributed its homoeologous genomes. METHODS: RNA-seq reads were obtained from the three species and were aligned against the genome sequence of Glycine max. Transcript levels were estimated for each gene, relative contributions of polyploidy-duplicated loci (homoeologues) in the tetraploid were identified, and comparisons of transcript profiles and individual genes were used to analyze the regulation of transcript levels. KEY RESULTS: We present a novel metric developed to address issues arising from high degrees of gene space duplication and a method for dissecting a gene's measured transcript level in a polyploid species into the relative contribution of its homoeologues. We identify the gene family likely contributing to differences in photosynthetic rate between the allotetraploid and its progenitors and show that the tetraploid appears to be using the "redundant" gene copies in novel ways. CONCLUSIONS: Given the prevalence of polyploidy events in plants, we believe many of the approaches developed here to be applicable, and often necessary, in most plant RNA-seq experiments. The deep sampling provided by RNA-seq allows us to dissect the genetic underpinnings of specific phenotypes as well as examine complex interactions within polyploid genomes.

Effects of elevated carbon dioxide on gas exchange and photochemical and nonphotochemical quenching at low temperature in tobacco plants varying in Rubisco activity.

Elevated (700 micromol mol-1) and ambient (350 micromol mol-1) CO2 effects on total ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activity, photosynthesis (A), and photoinhibition during 6 d at low temperature were measured on wild type (WT), and rbcS antisense DNA mutants (T3) of tobacco (Nicotiana tabacum L.) with 60% of WT total Rubisco activity. Prior to the low temperature treatment, A and quantum yield of PSII photochemistry in the light adapted state (phiPSII) were significantly lower in T3 compared to WT at each CO2 level. At this time, total nonphotochemical quenching (NPQTotal) levels were near maximal (0.75-0.85) in T3 compared to WT (0.39-0.50). A was stimulated by 107% in T3 and 25% in WT at elevated compared to ambient CO2. Pre-treatment acclimation to elevated CO2 occurred in WT resulting in lower Rubisco activity per unit leaf area and reduced stimulation of A. At low temperature, A of WT was similar at elevated and ambient CO2 while stimulation of A by elevated CO2 in T3 was reduced. In addition, at low temperature we measured significantly lower photochemical quenching at elevated CO2 compared to ambient CO2 in both genotypes. NPQTotal was similar (0.80-0.85) among all treatments. However, a larger proportion of NPQTotal was composed of qI,d, the damage subcomponent of the more slowly relaxing NPQ component, qI, in both genotypes at elevated compared to ambient CO2. Greater qI,d, at elevated CO2 during and after the low temperature treatment was not related to pre-treatment differences in total Rubisco activity.

Expression of complementary RNA from chloroplast transgenes affects editing efficiency of transgene and endogenous chloroplast transcripts.

The expression of angiosperm chloroplast genes is modified by C-to-U RNA editing. The mechanism for recognition of the approximately 30 C targets of editing is not understood. There is no single consensus sequence surrounding editing sites, though sites can be grouped into small 'clusters' of two to five sites exhibiting some sequence similarity. While complementary RNA that guides nucleotides for alteration has been detected in other RNA modification systems, it is not known whether complementary RNA is involved in chloroplast editing site recognition. We investigated the effect of expressing RNA antisense to the sequences -20 to +6 surrounding the RpoB-2 C target of editing, which is a member of a cluster that includes the PsbL-1 and Rps14-1 sites. Previous experiments had shown that chloroplast rpoB transgene transcripts carrying only these 27 nt were edited in vivo at the proper C. Though transcripts carrying sequences -31 to +60 surrounding the RpoB-2 sites were edited in chloroplast transgenic plants, transcripts carrying the -31 to +62 region followed by the 27 nt complementary region were not edited at all. In contrast, a similar construct, in which the C target as well as the preceding and subsequent nucleotides were mismatched within the 27 nt region, was efficiently edited. The presence of any of the four transgenes carrying RpoB-2 sequences in sense and/or antisense orientation resulted in reduced editing at the PsbL-1 site. Chloroplast transgenic plants expressing the three different antisense RNA constructs exhibited abnormal growth and development, though plants expressing the 92 nt sense transcripts were phenotypically normal.

Manipulation of root hair development and sorgoleone production in sorghum seedlings.

Sorghum (Sorghum bicolor) roots exude a potent bioherbicide sorgoleone. Previous work indicates that sorgoleone is produced in living root hairs. We have developed a mist system that resulted in abundant production of root hairs exuding sorgoleone and a mat system that significantly inhibited root hair development and consequently sorgoleone production. Applying Ag+ (an ethylene action inhibitor) at 1.2 mM to the seedlings grown in the mist system also inhibited root hair formation and elongation. Hypoxic conditions in the mist system did not result in the inhibition of root hair growth as compared to the standard air atmosphere (20.8% O2). Applying ethephon (an ethylene-releasing agent) at 0.031 mM to the roots of seedlings grown in the mat system with water running at 1 ml/min reversed the inhibition of root hair development by water movement. These results indicate that either water movement or ethylene can be utilized to manipulate root hair development and sorgoleone production in sorghum seedlings. It is hypothesized that water movement reduced the local ethylene concentration on the root surface and consequently inhibited root hair development of sorghum seedlings grown in the mat system.

Gas Exchange and Co-regulation of Photochemical and Nonphotochemical Quenching in Bean during Chilling at Ambient and Elevated Carbon Dioxide.

The effects of elevated (700 micromol mol(-1)) and ambient (350 micromol mol(-1)) CO(2) on gas exchange parameters and chlorophyll fluorescence were measured on bean (Phaseolus vulgaris) during 24 h chilling treatments at 6.5 degrees C. Consistent with previous research on this cultivar, photosynthetic decline during chilling was not significantly affected by CO(2) while post-chilling recovery was more rapid at elevated compared to ambient CO(2). Our primary focus was whether there were also CO(2)-mediated differences in demand on nonphotochemical quenching (NPQ) processes during the chilling treatments. We found that photosystem II quantum yield and total NPQ were similar between the CO(2) treatments during chilling. In both CO(2) treatments, chilling caused a shift from total NPQ largely composed of q(E), the protective, rapidly responding component of NPQ, to total NPQ dominated by the more slowly relaxing q(I), related to both protective and damage processes. The switch from q(E) to q(I) during chilling was more pronounced in the elevated CO(2) plants. Using complementary plots of the quantum yields of photochemistry and NPQ we demonstrate that, despite CO(2) effects on the partitioning of NPQ into q(E) and q(I) during chilling, total NPQ was regulated at both CO(2) levels to maximize photochemical utilization of absorbed light energy and dissipate only that fraction of light energy that was in excess of the capacity of photosynthesis. Photodamage did occur during chilling but was repaired within 3 h recovery from chilling in both CO(2) treatments.

Trehalose accumulation in rice plants confers high tolerance levels to different abiotic stresses.

Trehalose is a nonreducing disaccharide of glucose that functions as a compatible solute in the stabilization of biological structures under abiotic stress in bacteria, fungi, and invertebrates. With the notable exception of the desiccation-tolerant "resurrection plants," trehalose is not thought to accumulate to detectable levels in most plants. We report here the regulated overexpression of Escherichia coli trehalose biosynthetic genes (otsA and otsB) as a fusion gene for manipulating abiotic stress tolerance in rice. The fusion gene has the advantages of necessitating only a single transformation event and a higher net catalytic efficiency for trehalose formation. The expression of the transgene was under the control of either tissue-specific or stress-dependent promoters. Compared with nontransgenic rice, several independent transgenic lines exhibited sustained plant growth, less photo-oxidative damage, and more favorable mineral balance under salt, drought, and low-temperature stress conditions. Depending on growth conditions, the transgenic rice plants accumulate trehalose at levels 3-10 times that of the nontransgenic controls. The observation that peak trehalose levels remain well below 1 mgg fresh weight indicates that the primary effect of trehalose is not as a compatible solute. Rather, increased trehalose accumulation correlates with higher soluble carbohydrate levels and an elevated capacity for photosynthesis under both stress and nonstress conditions, consistent with a suggested role in modulating sugar sensing and carbohydrate metabolism. These findings demonstrate the feasibility of engineering rice for increased tolerance of abiotic stress and enhanced productivity through tissue-specific or stress-dependent overproduction of trehalose.