Balancing growth amidst salt stress - lifestyle perspectives from the extremophyte model Schrenkiella parvula.

Schrenkiella parvula, a leading extremophyte model in Brassicaceae, can grow and complete its lifecycle under multiple environmental stresses, including high salinity. Yet, the key physiological and structural traits underlying its stress-adapted lifestyle are unknown along with trade-offs when surviving salt stress at the expense of growth and reproduction. We aimed to identify the influential adaptive trait responses that lead to stress-resilient and uncompromised growth across developmental stages when treated with salt at levels known to inhibit growth in Arabidopsis and most crops. Its resilient growth was promoted by traits that synergistically allowed primary root growth in seedlings, the expansion of xylem vessels across the root-shoot continuum, and a high capacity to maintain tissue water levels by developing thicker succulent leaves while enabling photosynthesis during salt stress. A successful transition from vegetative to reproductive phase was initiated by salt-induced early flowering, resulting in viable seeds. Self-fertilization in salt-induced early flowering was dependent upon filament elongation in flowers otherwise aborted in the absence of salt during comparable plant ages. The maintenance of leaf water status promoting growth, and early flowering to ensure reproductive success in a changing environment, were among the most influential traits that contributed to the extremophytic lifestyle of S. parvula.

Living with high potassium: Balance between nutrient acquisition and K-induced salt stress signaling.

High potassium (K) in the growth medium induces salinity stress in plants. However, the molecular mechanisms underlying plant responses to K-induced salt stress are virtually unknown. We examined Arabidopsis (Arabidopsis thaliana) and its extremophyte relative Schrenkiella parvula using a comparative multiomics approach to identify cellular processes affected by excess K and understand which deterministic regulatory pathways are active to avoid tissue damages while sustaining growth. Arabidopsis showed limited capacity to curb excess K accumulation and prevent nutrient depletion, contrasting to S. parvula which could limit excess K accumulation without restricting nutrient uptake. A targeted transcriptomic response in S. parvula promoted nitrogen uptake along with other key nutrients followed by uninterrupted N assimilation into primary metabolites during excess K-stress. This resulted in larger antioxidant and osmolyte pools and corresponded with sustained growth in S. parvula. Antithetically, Arabidopsis showed increased reactive oxygen species levels, reduced photosynthesis, and transcriptional responses indicative of a poor balance between stress signaling, subsequently leading to growth limitations. Our results indicate that the ability to regulate independent nutrient uptake and a coordinated transcriptomic response to avoid nonspecific stress signaling are two main deterministic steps toward building stress resilience to excess K+-induced salt stress.

Arabidopsis plastid carbonic anhydrase ßCA5 is important for normal plant growth.

Carbonic anhydrases (CAs) are zinc-metalloenzymes that catalyze the interconversion of CO2 and HCO3-. In heterotrophic organisms, CAs provide HCO3- for metabolic pathways requiring a carboxylation step. Arabidopsis (Arabidopsis thaliana) has 14 α- and ß-type CAs, two of which are plastid CAs designated as ßCA1 and ßCA5. To study their physiological properties, we obtained knock-out (KO) lines for ßCA1 (SALK_106570) and ßCA5 (SALK_121932). These mutant lines were confirmed by genomic PCR, RT-PCR, and immunoblotting. While ßca1 KO plants grew normally, growth of ßca5 KO plants was stunted under ambient CO2 conditions of 400 µL L-1; high CO2 conditions (30,000 µL L-1) partially rescued their growth. These results were surprising, as ßCA1 is more abundant than ßCA5 in leaves. However, tissue expression patterns of these genes indicated that ßCA1 is expressed only in shoot tissue, while ßCA5 is expressed throughout the plant. We hypothesize that ßCA5 compensates for loss of ßCA1 but, owing to its expression being limited to leaves, ßCA1 cannot compensate for loss of ßCA5. We also demonstrate that ßCA5 supplies HCO3- required for anaplerotic pathways that take place in plastids, such as fatty acid biosynthesis.

The Cytoplasmic Carbonic Anhydrases ßCA2 and ßCA4 Are Required for Optimal Plant Growth at Low CO2.

Carbonic anhydrases (CAs) are zinc metalloenzymes that interconvert CO2 and HCO3 (-) In plants, both α- and ß-type CAs are present. We hypothesize that cytoplasmic ßCAs are required to modulate inorganic carbon forms needed in leaf cells for carbon-requiring reactions such as photosynthesis and amino acid biosynthesis. In this report, we present evidence that ßCA2 and ßCA4 are the two most abundant cytoplasmic CAs in Arabidopsis (Arabidopsis thaliana) leaves. Previously, ßCA4 was reported to be localized to the plasma membrane, but here, we show that two forms of ßCA4 are expressed in a tissue-specific manner and that the two proteins encoded by ßCA4 localize to two different regions of the cell. Comparing transfer DNA knockout lines with wild-type plants, there was no reduction in the growth rates of the single mutants, ßca2 and ßca4 However, the growth rate of the double mutant, ßca2ßca4, was reduced significantly when grown at 200 µL L(-1) CO2 The reduction in growth of the double mutant was not linked to a reduction in photosynthetic rate. The amino acid content of leaves from the double mutant showed marked reduction in aspartate when compared with the wild type and the single mutants. This suggests the cytoplasmic CAs play an important but not previously appreciated role in amino acid biosynthesis.

The carbon concentrating mechanism in Chlamydomonas reinhardtii: finding the missing pieces.

The photosynthetic, unicellular green alga, Chlamydomonas reinhardtii, lives in environments that often contain low concentrations of CO2 and HCO3 (-), the utilizable forms of inorganic carbon (Ci). C. reinhardtii possesses a carbon concentrating mechanism (CCM) which can provide suitable amounts of Ci for growth and development. This CCM is induced when the CO2 concentration is at air levels or lower and is comprised of a set of proteins that allow the efficient uptake of Ci into the cell as well as its directed transport to the site where Rubisco fixes CO2 into biomolecules. While several components of the CCM have been identified in recent years, the picture is still far from complete. To further improve our knowledge of the CCM, we undertook a mutagenesis project where an antibiotic resistance cassette was randomly inserted into the C. reinhardtii genome resulting in the generation of 22,000 mutants. The mutant collection was screened using both a published PCR-based approach (Gonzalez-Ballester et al. 2011) and a phenotypic growth screen. The PCR-based screen did not rely on a colony having an altered growth phenotype and was used to identify colonies with disruptions in genes previously identified as being associated with the CCM-related gene. Eleven independent insertional mutations were identified in eight different genes showing the usefulness of this approach in generating mutations in CCM-related genes of interest as well as identifying new CCM components. Further improvements of this method are also discussed.

Carbonic anhydrases in the cell wall and plasma membrane of Arabidopsis thaliana are required for optimal plant growth on low CO2.

Introduction: Plants have many genes encoding both alpha and beta type carbonic anhydrases. Arabidopsis has eight alpha type and six beta type carbonic anhydrase genes. Individual carbonic anhydrases are localized to specific compartments within the plant cell. In this study, we investigate the roles of αCA2 and ßCA4.1 in the growth of the plant Arabidopsis thaliana under different CO2 regimes. Methods: Here, we identified the intracellular location of αCA2 and ßCA4.1 by linking the coding region of each gene to a fluorescent tag. Tissue expression was determined by investigating GUS expression driven by the αCA2 and ßCA4.1 promoters. Finally, the role of these proteins in plant growth and photosynthesis was tested in plants with T-DNA insertions in the αCA2 and ßCA4 genes. Results: Fluorescently tagged proteins showed that αCA2 is localized to the cell wall and ßCA4.1 to the plasma membrane in plant leaves. Both proteins were expressed in roots and shoots. Plants missing either αCA2 or ßCA4 did not show any growth defects under the conditions tested in this study. However, if both αCA2 and ßCA4 were disrupted, plants had a significantly smaller above- ground fresh weight and rosette area than Wild Type (WT) plants when grown at 200 µL L-1 CO2 but not at 400 and 1,000 µL L-1 CO2. Growth of the double mutant plants at 200 µL L-1 CO2 was restoredif either αCA2 or ßCA4.1 was transformed back into the double mutant plants. Discussion: Both the cell wall and plasma membrane CAs, αCA2 and ßCA4.1 had to be knocked down to produce an effect on Arabidopsis growth and only when grown in a CO2 concentration that was significantly below ambient. This indicates that αCA2 and ßCA4.1 have overlapping functions since the growth of lines where only one of these CAs was knocked down was indistinguishable from WT growth. The growth results and cellular locations of the two CAs suggest that together, αCA2 and ßCA4.1 play an important role in the delivery of CO2 and HCO3 - to the plant cell.

Photorespiration and carbon concentrating mechanisms: two adaptations to high O2, low CO2 conditions.

This review presents an overview of the two ways that cyanobacteria, algae, and plants have adapted to high O2 and low CO2 concentrations in the environment. First, the process of photorespiration enables photosynthetic organisms to recycle phosphoglycolate formed by the oxygenase reaction catalyzed by ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). Second, there are a number of carbon concentrating mechanisms that increase the CO2 concentration around Rubisco which increases the carboxylase reaction enhancing CO2 fixation. This review also presents possibilities for the beneficial modification of these processes with the goal of improving future crop yields.