Salt stress is an ever-increasing stressor that affects both plants and humans. Therefore, developing strategies to limit the undesirable effects of salt stress is essential. Sodium ion exclusion is well known for its efficient salt-tolerance mechanism. The High-affinity K+ Transporter (HKT) excludes excess Na+ from the transpiration stream. This study identified and characterized the HKT protein family in Bienertia sinuspersici, a single-cell C4 plant. The HKT and Salt Overly Sensitive 1 (SOS1) expression levels were examined in B. sinuspersici and Arabidopsis thaliana leaves under four different salt stress conditions: 0, 100, 200, and 300 mM NaCl. Furthermore, BsHKT1;2 was cloned, thereby producing stable transgenic Brassica rapa. Our results showed that, compared to A. thaliana as a glycophyte, the HKT family is expanded in B. sinuspersici as a halophyte with three paralogs. The phylogenetic analysis revealed three paralogs belonging to the HKT subfamily I. Out of three copies, the expression of BsHKT1;2 was higher in Bienertia under control and salt stress conditions than in A. thaliana. Stable transgenic plants overexpressing 35S::BsHKT1;2 showed higher salt tolerance than non-transgenic plants. Higher biomass and longer roots were observed in the transgenic plants under salt stress than in non-transgenic plants. This study demonstrates the evolutionary and functional differences in HKT proteins between glycophytes and halophytes and associates the role of BsHKT1;2 in imparting salt tolerance and productivity.

Single-cell C4 photosynthesis (SCC4) in terrestrial plants without Kranz anatomy involves three steps: initial CO2 fixation in the cytosol, CO2 release in mitochondria, and a second CO2 fixation in central chloroplasts. Here, we investigated how the large number of mechanisms underlying these processes, which occur in three different compartments, are orchestrated in a coordinated manner to establish the C4 pathway in Bienertia sinuspersici, a SCC4 plant. Leaves were subjected to transcriptome analysis at three different developmental stages. Functional enrichment analysis revealed that SCC4 cycle genes are coexpressed with genes regulating cyclic electron flow and amino/organic acid metabolism, two key processes required for the production of energy molecules in C3 plants. Comparative gene expression profiling of B. sinuspersici and three other species (Suaeda aralocaspica, Amaranthus hypochondriacus, and Arabidopsis thaliana) showed that the direction of metabolic flux was determined via an alteration in energy supply in peripheral chloroplasts and mitochondria via regulation of gene expression in the direction of the C4 cycle. Based on these results, we propose that the redox homeostasis of energy molecules via energy metabolism regulation is key to the establishment of the SCC4 pathway in B. sinuspersici.

Single-cell C4 (SCC4) plants with bienertioid anatomy carry out photosynthesis in a single cell. Chloroplast movement is the underlying phenomenon, where chloroplast unusual positioning 1 (CHUP1) plays a key role. This study aimed to characterize CHUP1 and CHUP1-like proteins in an SCC4 photosynthetic plant, Bienertia sinuspersici. Also, a comparative analysis of SCC4 CHUP1 was made with C3, C4, and CAM model plants including an extant basal angiosperm, Amborella. The CHUP1 gene exists as a single copy from the basal angiosperms to SCC4 plants. Our analysis identified that Chenopodium quinoa, a recently duplicated allotetraploid, has two copies of CHUP1. In addition, the numbers of CHUP1-like and its associated proteins such as CHUP1-like_a, CHUP1-like_b, HPR, TPR, and ABP varied between the species. Hidden Markov Model analysis showed that the gene size of CHUP1-like_a and CHUP1-like_b of SCC4 species, Bienertia, and Suaeda were enlarged than other plants. Also, we identified that CHUP1-like_a and CHUP1-like_b are absent in Arabidopsis and Amborella, respectively. Motif analysis identified several conserved and variable motifs based on the orders (monocot and dicot) as well as photosynthetic pathways. For instance, CAM plants such as pineapple and cactus shared certain motifs of CHUP1-like_a irrespective of their distant phylogenetic relationship. The free ratio model showed that CHUP1 maintained purifying selection, whereas CHUP1-like_a and CHUP1-like_b have adaptive functions between SCC4 plants and quinoa. Similarly, rice and maize branches displayed functional diversification on CHUP1-like_b. Relative gene expression data showed that during the subcellular compartmentalization process of Bienertia, CHUP1 and actin-binding proteins (ABP) genes showed a similar pattern of expression. Altogether, the results of this study provide insight into the evolutionary and functional details of CHUP1 and its associated proteins in the development of the SCC4 system in comparison with other C3, C4, and CAM model plants.

Single cell C4 (SCC4) plants, discovered around two decades ago, are promising materials for efforts for genetic engineering of C4 photosynthesis into C3 crops. Unlike C4 plants with Kranz anatomy, they exhibit a fully functional C4 photosynthesis in just a single cell and do not require mesophyll and bundle sheath cell spatial separation. Bienertia sinuspersici is one such SCC4 plant, with NAD-malic enzyme (NAD-ME) subtype C4 photosynthesis. Its chlorenchyma cell consist of two compartments, peripheral compartment (PC), analogous to mesophyll cell, and central compartment (CC), analogous to bundle sheath cell. Since oxidative stress creates an important constraint for plants under salinity and drought, we comparatively examined the response of enzymatic antioxidant system, H2O2 and TBARS contents, peroxiredoxin Q, NADPH thioredoxin reductase C, and plastid terminal oxidase protein levels of PC chloroplasts (PCC) and CC chloroplasts (CCC). Except for protein levels, these parameters were also examined on the whole leaf level, as well as catalase and NADPH oxidase activities, water status and growth parameters, and levels of C4 photosynthesis related transcripts. Many C4 photosynthesis related transcript levels were elevated, especially under drought. Activities of dehydroascorbate reductase and especially peroxidase were elevated under drought in both compartments (CCC and PCC). Even though decreases of antioxidant enzyme activities were more prevalent in PCC, and the examined redox regulating protein levels, especially of peroxiredoxin Q, were elevated in CCC under both stresses, PCC was less damaged by either stress. These suggest PCC is more tolerant and has other means of preventing or alleviating oxidative damage.

The Bienertia sinuspersici chloroplast whole genome (cpDNA) sequencing was completed in this study. Bienertia sinuspersici chloroplast genome is 153,472 bp in length and contains 127 genes, such as 83 unique protein-coding genes, 36 tRNA genes and eight rRNA genes. There were two inverted repeat regions (IR) and small and large single-copy regions (SSC and LSC) with 24,948 bp, 19,016 and 84,560 bp, respectively. 59% of the B. sinuspersici cpDNA consisted of gene-coding regions (protein-coding and RNA genes). The overall GC contents of the B. sinuspersici cpDNA were 36.59% and in the LSC, SSC and IR regions were 34.47%, 29.42% and 42.94%, respectively. A phylogenetic analysis of seven complete cpDNA from Chenopodiaceae family shows that B. sinuspersici cpDNA is closely related to Salicornia species.

C4 plants are efficient in suppressing photorespiration and enhancing carbon gain as compared to C3 plants. Bienertia sinuspersici Akhani is one of the few species in the family Amaranthaceae that can perform C4 photosynthesis within individual chlorenchyma cells, without the conventional Kranz anatomy in its leaf. This plant is salt-tolerant and is well-adapted to thrive in hot and humid climates. To date, there have been no reported cytogenetic analyses yet on this species.
This study aims to provide a cytogenetic analysis of B. sinuspersici as the first step in genome sequencing.
Fluorescence in situ hybridization (FISH) karyotype analysis was conducted using the metaphase chromosomes of B. sinuspersici probed with 5S and 45S rDNA and Arabidopsis-type telomeric repeats.
Results of the cytogenetic analysis confirmed that B. sinuspersici has 2n = 2x = 18 consisting of nine pairs of metacentric chromosomes. Two loci of 45S rDNA were found on the distal regions of the short arm of chromosome 7. Nine loci of 5S rDNA were found in the pericentromeric regions of chromosomes 1, 3, 4, 6, and 8, which also colocalized with Arabidopsis-type telomeric repeats; while four loci in the interstitial regions of chromosome 5 and 8 can be observed. The single locus of 5S rDNA that was found in chromosome 8 appears to be hemizygous.
The FISH karyotype analysis, based on the combination of rDNAs, telomeric tandem repeat markers and C0t DNA chromosome landmarks, allowed efficient chromosome identification and provided useful information in characterizing the genome of B. sinuspersici.

The emergence and expression of the YABBY gene family (YGF) coincided with the evolution of leaves in seed plants, and was integral to the early evidence of lamina followed by reproductive development. YGF contains six subclasses, i.e., CRC, INO, FIL, YAB2, YAB3, and YAB5. This study aims to extract the genome sequences of the YGF in Bienertia sinuspersici, an important model plant for single-cell C4 (SCC4), non-Kranz photosynthesis. A comparative genomic analysis was undertaken with Vitis vinefera, Arabidopsis thaliana, Brassica rapa, and Chenopodium quinoa. Six copies of YGF were present in B. sinuspersici and A. thaliana with a single copy of each YGF subgroup. V. vinefera possessed seven copies of YGF with duplicates in FIL and YAB2 subgroups, but no YAB3. B. rapa and C. quinoa after whole genome duplication contained additional copies of YGF. The gene structure and conserved motifs were analyzed among the YGF. In addition, the relative quantification of YGF was analyzed in the leaves, reproductive developmental stages such as the bud, and the pre-anthesis and anthesis stages in B. sinuspersici, A. thaliana, and B. rapa. CRC and INO possessed conserved floral-specific expression. Temporal and perpetual changes in the expression of YGF orthologs were observed in the leaves and reproductive developmental stages. The results of this study provide an overview of YGF evolution, copy number, and its differential expression in B. sinuspersici. Further studies are required to shed light on the roles of YABBY genes in the evolution of SCC4 plants and their distinct physiologies.

The identification of novel herbicides is of crucial importance to modern agriculture. We developed an efficient in vivo assay based on oxygen evolution measurements using suspensions of chlorenchyma cells isolated from the single-cell C4 plant Bienertia sinuspersici to identify and characterize inhibitors of C4 photosynthesis. This novel approach fills the gap between conventional in vitro assays for inhibitors targeting C4 key enzymes and in vivo experiments on whole plants. The assay addresses inhibition of the target enzymes in a plant context thereby taking care of any reduced target inhibition due to metabolization or inadequate uptake of small molecule inhibitors across plant cell walls and membranes. Known small molecule inhibitors targeting C4 photosynthesis were used to validate the approach. To this end, we tested pyruvate phosphate dikinase inhibitor bisindolylmaleimide IV and phosphoenolpyruvate carboxylase inhibitor okanin. Both inhibitors show inhibition of plant photosynthesis at half-maximal inhibitory concentrations in the sub-mM range and confirm their potential to act as a new class of C4 selective inhibitors.

Bienertia sinuspersici performs single cell C4 photosynthesis without Kranz anatomy. Peripheral and central cytoplasmic compartments in a single chlorenchyma cell act as mesophyll cells and bundle sheath cells. Development of this specialized mechanism is gradual during plant development. Young leaves perform C3 photosynthesis, while mature leaves have complete C4 cycle. The aim of this work was to investigate changes in redox regulation and antioxidant defence during transition from C3 to single cell C4 photosynthesis in B. sinuspersici leaves. First, we confirmed gradual development of C4 with protein blot and qRT-PCR analysis of C4 enzymes. After this activities and isoenzymes of superoxide dismutase (SOD), catalase (CAT), peroxidase (POX), ascorbate peroxidase (APX), glutathione reductase (GR), dehydroascorbate reductase (DHAR) and H2O2 and TBARS and glutathione pool and redox status (GSH/GSSG) were determined in young, developing and mature leaves during transition from C3 to single cell C4 photosynthesis. Activities of SOD, APX and POX decrease, while GR and DHAR were increased. However, most striking results were the changes in isoenzyme patterns of SOD, CAT and GR which were gradual through transition to C4 photosynthesis.

Temporal and spatial patterns of photosynthetic enzyme expression and structural maturation of chlorenchyma cells along longitudinal developmental gradients were characterized in young leaves of two single cell C4 species, Bienertia sinuspersici and Suaeda aralocaspica Both species partition photosynthetic functions between distinct intracellular domains. In the C4-C domain, C4 acids are formed in the C4 cycle during capture of atmospheric CO2 by phosphoenolpyruvate carboxylase. In the C4-D domain, CO2 released in the C4 cycle via mitochondrial NAD-malic enzyme is refixed by Rubisco. Despite striking differences in origin and intracellular positioning of domains, these species show strong convergence in C4 developmental patterns. Both progress through a gradual developmental transition towards full C4 photosynthesis, with an associated increase in levels of photosynthetic enzymes. Analysis of longitudinal sections showed undeveloped domains at the leaf base, with Rubisco rbcL mRNA and protein contained within all chloroplasts. The two domains were first distinguishable in chlorenchyma cells at the leaf mid-regions, but still contained structurally similar chloroplasts with equivalent amounts of rbcL mRNA and protein; while mitochondria had become confined to just one domain (proto-C4-D). The C4 state was fully formed towards the leaf tips, Rubisco transcripts and protein were compartmentalized specifically to structurally distinct chloroplasts in the C4-D domains indicating selective regulation of Rubisco expression may occur by control of transcription or stability of rbcL mRNA. Determination of CO2 compensation points showed young leaves were not functionally C4, consistent with cytological observations of the developmental progression from C3 default to intermediate to C4 photosynthesis.