Isaiah Catalino M. Pabuayon

The physiological role of chloroplast carbonic anhydrases (CAs) has long been debated, particularly in the context of photosynthesis. While early hypotheses proposed that CAs enhance CO2 assimilation by rapidly accessing the HCO3− pool, direct evidence has been lacking. In this study, we examined Arabidopsis (Arabidopsis thaliana) mutants lacking both chloroplast-localized βCA1 (AT3G01500) and βCA5 (AT4G33580) to assess their impact on plant growth and photosynthetic performance. Our results show that plants deficient in chloroplast CA activity are unable to grow under ambient CO2 conditions (400 μL L⁻1) but can complete their life cycle under elevated CO2 levels (≥12,000 μL L⁻1). However, CO2 assimilation rates and ΦII measurements in CA-deficient plants were comparable to those in wild-type plants under 0.04% (400 μL L⁻1), 0.4% (4,000 μL L⁻1), and 4% CO2 (40,000 μL L⁻1) concentrations, indicating that chloroplast CAs are not essential for photosynthetic CO2 fixation. Instead, our findings suggest that chloroplast CA activity is critical for supporting other metabolic pathways, namely amino acid, nucleic acid, and fatty acid biosynthesis. Expression of the Chlamydomonas (Chlamydomonas reinhardtii) bicarbonate transporter LCIA in chloroplast CA mutants partially rescued the growth phenotype under near-ambient CO2 conditions. These LCIA-complemented lines showed no difference in photosynthesis, further supporting the role of CAs in non-photosynthetic reactions. This work provides direct evidence that while chloroplast CAs are dispensable for photosynthesis, they are essential for plant growth and development under ambient CO2 due to their role in increasing the bicarbonate concentration for specific anaplerotic pathways.

Understanding the nature of non-parental phenotypes created by transgressive segregation is important in creating novel genetic recombinants that can withstand different environmental conditions for crop production. FL510, a transgressive salinity-tolerant rice genotype from a cross between IR29 (salt-sensitive) and Pokkali (salt-tolerant), has tolerance mechanisms active under control conditions and improves survival upon the onset of salinity. This study compares normal-state metabolomes and lipidomes of FL510 with its parents. Principal component analysis (PCA) of the identified analytes showed clear and expected similarity between FL510 and Pokkali, while partial least squares discriminant analysis (PLS-DA) emphasized overlaps between the metabolic profiles of IR29 and FL510. The analysis identified metabolites with inherited patterns of abundance from either parent in FL510 and those with unique, non-parental abundances, and these were supported by differential expression of key pathway-related genes identified through transcriptome analysis. Strigolactone precursor production was identified as a key feature in FL510, which may help explain its unique architecture that is beneficial for osmotic stress. We also identified a divergence between productivity under ideal environments leading to free radical production versus tempered production that offers better survival under marginal growing conditions. FL510 showed an inheritance of hormone and amino acid abundances from Pokkali, which further explains some of its architectural and previously studied stress-response features. Meanwhile, the similarity of FL510 with IR29 in terms of flavonoid indicates an inheritance of productivity and is consistent with previous reports of induction for these molecules under stress, rather than being active under control conditions. Main Conclusion: Through repeated genetic recombination of genetically distant alleles, the transgressive segregant FL510 gained unique, non-parental signaling pathways and complementary metabolome features from both parents leading to positive net genetic gains.

The ATP-driven bicarbonate transporter 1 (BCT1) from Synechococcus is a four-component complex in the cyanobacterial CO₂-concentrating mechanism. BCT1 could enhance photosynthetic CO₂ assimilation in plant chloroplasts. However, directing its subunits (CmpA, CmpB, CmpC, and CmpD) to three chloroplast sub-compartments is highly complex. Investigating BCT1 integration into Nicotiana benthamiana chloroplasts revealed promising targeting strategies using transit peptides from the intermembrane space protein Tic22 for correct CmpA targeting, while the transit peptide of the chloroplastic ABCD2 transporter effectively targeted CmpB to the inner envelope membrane. CmpC and CmpD were targeted to the stroma by RecA and recruited to the inner envelope membrane by CmpB. Despite successful targeting, expression of this complex in CO₂-dependent Escherichia coli failed to demonstrate bicarbonate uptake. We then used rational design and directed evolution to generate new BCT1 forms that were constitutively active. Several mutants were recovered, including a CmpCD fusion. Selected mutants were further characterized and stably expressed in Arabidopsis thaliana, but the transformed plants did not have higher carbon assimilation rates or decreased CO₂ compensation points in mature leaves. While further analysis is required, this directed evolution and heterologous testing approach presents potential for iterative modification and assessment of CO₂-concentrating mechanism components to improve plant photosynthesis.

The ATP-driven bicarbonate transporter 1 (BCT1), a four-component complex in the cyanobacteria CO ₂ concentrating mechanism, could enhance photosynthetic CO ₂ assimilation in plant chloroplasts. However, directing its subunits (CmpA, CmpB, CmpC and CmpD) to three chloroplast sub-compartments is highly complex. Investigating BCT1 integration into Nicotiana benthamiana chloroplasts revealed promising targeting strategies using transit peptides from the intermembrane space protein Tic22 for correct CmpA targeting, while the transit peptide of the chloroplastic ABCD2 transporter effectively targeted CmpB to the inner envelope membrane. CmpC and CmpD were targeted to the stroma by RecA and recruited to the inner envelope membrane by CmpB. Despite successful targeting, expression of this complex in CO ₂ -dependent Escherichia coli failed to demonstrate bicarbonate uptake. We then used rational design and directed evolution to generate new BCT1 forms that were constitutively active. Several mutants were recovered, including a CmpCD fusion. Selected mutants were further characterized and stably expressed in Arabidopsis thaliana , but the transformed plants did not have higher carbon assimilation rates or decreased CO ₂ compensation points in mature leaves. While further analysis is required, this directed evolution and heterologous testing approach presents potential for iterative modification and assessment of CO ₂ concentrating mechanism components to improve plant photosynthesis.

The ratio of Na⁺ and K⁺ is an important determinant of the magnitude of Na⁺ toxicity and osmotic stress in plant cells. Traditional analytical approaches involve destructive tissue sampling and chemical analysis, where real-time observation of spatio-temporal experiments across genetic or breeding populations is unrealistic. Such an approach can also be very inaccurate and prone to erroneous biological interpretation. Analysis by Hyperspectral Imaging (HSI) is an emerging non-destructive alternative for tracking plant nutrient status in a time-course with higher accuracy and reduced cost for chemical analysis. In this study, the feasibility and predictive power of HSI-based approach for spatio-temporal tracking of Na⁺ and K⁺ levels in tissue samples was explored using a panel recombinant inbred line (RIL) of rice (Oryza sativa L.; salt-sensitive IR29 x salt-tolerant Pokkali) with differential activities of the Na⁺ exclusion mechanism conferred by the SalTol QTL. In this panel of RILs the spectrum of salinity tolerance was represented by FL499 (super-sensitive), FL454 (sensitive), FL478 (tolerant), and FL510 (super-tolerant). Whole-plant image processing pipeline was optimized to generate HSI spectra during salinity stress at EC = 9 dS m^-1. Spectral data was used to create models for Na⁺ and K⁺ prediction by partial least squares regression (PLSR). Three datasets, i.e., mean image pixel spectra, smoothened version of mean image pixel spectra, and wavelength bands, with wide differences in intensity between control and salinity facilitated the prediction models with high R². The smoothened and filtered datasets showed significant improvements over the mean image pixel dataset. However, model prediction was not fully consistent with the empirical data. While the outcome of modeling-based prediction showed a great potential for improving the throughput capacity for salinity stress phenotyping, additional technical refinements including tissue-specific measurements is necessary to maximize the accuracy of prediction models.

Introgression of major-effect QTLs is an important component of rice breeding for yield-retention under drought. While largely effective, the maximum potentials of such QTLs have not been consistent across genetic backgrounds. We hypothesized that synergism or antagonism with additive-effect peripheral genes across the background could either enhance or undermine the QTL effects. To elucidate the molecular underpinnings of such interaction, we dissected qDTY12.1 synergy with numerous peripheral genes in context of network rewiring effects. By integrative transcriptome profiling and network modeling, we identified the DECUSSATE ( OsDEC ) within qDTY12.1 as the core of the synergy and shared by two sibling introgression lines in IR64 genetic background, i.e., LPB (low-yield penalty) and HPB (high-yield penalty). OsDEC is expressed in flag leaves and induced by progressive drought at booting stage in LPB but not in HPB. The unique OsDEC signature in LPB is coordinated with 35 upstream and downstream peripheral genes involved in floral development through the cytokinin signaling pathway, which are lacking in HPB. Results further support the differential network rewiring effects through genetic coupling-uncoupling between qDTY12.1 and other upstream and downstream peripheral genes across the distinct genetic backgrounds of LPB and HPB. We propose that the functional DEC -network in LPB defines a mechanism for early flowering as a means for avoiding the depletion of photosyntate needed for reproductive growth due to drought. Its impact on yield-retention is likely through the timely establishment of stronger source-sink dynamics that sustains a robust reproductive transition under drought. Author summary: While the Green Revolution of the 1960’s significantly increased rice grain yields through the creation of high-yielding varieties for high input systems, current marginal climates pose a significant challenge for providing consistent yield. In rice growing regions of the world, drought affects the livelihood of small-scale and subsistence farmers by inflicting significant yield penalties to their production systems. Breeding of next-generation rice varieties with optimal balance of survivability and productivity traits will be key to providing consistent yields year to year. Within this paradigm, the use of large effect QTLs such as qDTY12.1 to improve yield retention under drought have been largely successful. By integrating the use of high resolution transcriptome datasets with a focused biological interrogation of agronomic results from this and previous studies, we uncovered a putative functional genetic network, anchored by the DECUSSATE gene ( OsDEC ) within qDTY12.1 , that effectively minimizes drought penalties to yield by driving cellular processes that culminate in timely flowering that maximizes the use of photosynthetic sources for efficient reproduductive transition and ultimately seed development. Our study further illuminates the qDTY12.1 function and speaks to the misconception that qDTY introgression alone is sufficient for providing consistently large positive effects to yield retention under reproductive stage drought.