Sweet potato (Ipomoea batatas L.) genotype selection using advanced indices and statistical models: A multi-year approach.

In Bangladesh, sweet potato holds the fourth position as a crucial carbohydrate source, trailing rice, wheat, and potato. However, locally grown sweet potato varieties often display limited stability and yield. To tackle this challenge, diverse selection methods and statistical models were utilized to pinpoint sweet potato genotypes showcasing both stability and superior yield and quality traits. In the initial two years, multiple selection methods were employed to narrow down the collections based on preferences for yield and its contributing traits. Subsequently, a multi-environment trial (MET) was conducted in the following year to pinpoint superior and stable genotypes with desirable yield and quality characteristics. An integrated approach involving the Multi-Trait Genotype Ideotype Distance Index (MGIDI), Factor Analysis and Ideotype-Design (FAI-BLUP), and Smith-Hazel Index (SH) led to the identification of 71 superior sweet potato genotypes out of a total of 351 in the initial growing season. In the subsequent season, the MGIDI selection index was applied to the 71 genotypes, resulting in the selection of 11 top-performing genotypes. This selection process was complemented by a detailed analysis of the strengths and weaknesses of the selected genotypes. In the MET, the mixed effect model, specifically the linear mixed model (LMM), identified significant genotypic and genotype-environment interaction (GEI) variances. This points to elevated heritability and selection accuracy, ultimately boosting the model's reliability. By combining the strengths of LMM and additive main effects and multiplicative interaction (AMMI), the best linear unbiased prediction (BLUP) index identified H20 as the top-performing genotype for marketable root yield (MRY), H37 for dry weight of root (DW), H8 for beta carotene (BC) and H41 for vitamin c (VC). These genotypes surpassed the overall average in the WAAS index. For simultaneous stability and high performance, the WAASBY index selected H37 for MRY, H6 for DW, H61 for BC, and H3 for VC. Finally, genotypes H3 and H20 were selected using multi-trait stability index (MTSI), as they possessed high performance and stability. Based on the selection sense, the objective has been achieved with regards to the trait MRW, which serves as a major criterion for a superior variety of sweet potato.

Distance-Based Phylogenetic Placement with Statistical Support.

Phylogenetic identification of unknown sequences by placing them on a tree is routinely attempted in modern ecological studies. Such placements are often obtained from incomplete and noisy data, making it essential to augment the results with some notion of uncertainty. While the standard likelihood-based methods designed for placement naturally provide such measures of uncertainty, the newer and more scalable distance-based methods lack this crucial feature. Here, we adopt several parametric and nonparametric sampling methods for measuring the support of phylogenetic placements that have been obtained with the use of distances. Comparing the alternative strategies, we conclude that nonparametric bootstrapping is more accurate than the alternatives. We go on to show how bootstrapping can be performed efficiently using a linear algebraic formulation that makes it up to 30 times faster and implement this optimized version as part of the distance-based placement software APPLES. By examining a wide range of applications, we show that the relative accuracy of maximum likelihood (ML) support values as compared to distance-based methods depends on the application and the dataset. ML is advantageous for fragmentary queries, while distance-based support values are more accurate for full-length and multi-gene datasets. With the quantification of uncertainty, our work fills a crucial gap that prevents the broader adoption of distance-based placement tools.

Histopathology of head kidney tissues in challenged rohu, Labeo rohita Hamilton after vaccinating with Aeromonas hydrophila antigens.

The study was conducted to evaluate the vaccination effects on rohu, Labeo rohita head kidney tissues while assessing the vaccine efficacy of Aeromonas hydrophila antigens. Six acclimatized rohu groups were immunized with three antigenic formulations (outer membrane proteins, somatic and whole-cell antigen) @ 200 µg/fish and also with equal volume of Freund's incomplete adjuvant (FIA), separately. Simultaneously, two non-vaccinated groups, i.e., injected with FIA (100µl), normal saline solutions (0.85%) and one control without injection were maintained for 28 days. All rohu were challenged with median lethal dose of A. hydrophila (2.85 × 106 cells/rohu) intraperitoneally. After 7 days, highest cumulative mortality (%) of ˃88% was found for all non-vaccinated groups. During histopathological observations in head kidney tissues of all treatment and control groups, numerous histopathological changes in the nephritic cells like mild loss of typical tubular epithelial lining, necrosis, thickening of renal epithelial lining, haemorrhages, inflammation, distorted and widening of the lumen with vacuolated surrounding and the constricted lumen of nephritic tubules were noticed for vaccinated rohu in contrast to non vaccinated groups before A. hydrophila challenge. In case of all non-vaccinated fish, including control, extensive degenerated and necrotized head kidney tissues were observed, whereas it was least observed in vaccinated rohu after 7 days A. hydrophila challenge. Results suggest that OMPs antigen along with FIA was the premier vaccine approach for improving resistance to Aeromonas disease and reduce mortality in rohu. Similarly, vaccination with all three antigenic formulations, preferably when applied along with FIA, can effectively protect the head kidney against A. hydrophila infection.

The roles of the chaperone-like protein CpeZ and the phycoerythrobilin lyase CpeY in phycoerythrin biogenesis.

Phycoerythrin (PE) present in the distal ends of light-harvesting phycobilisome rods in Fremyella diplosiphon (Tolypothrix sp. PCC 7601) contains five phycoerythrobilin (PEB) chromophores attached to six cysteine residues for efficient green light capture for photosynthesis. Chromophore ligation on PE subunits occurs through bilin lyase catalyzed reactions, but the characterization of the roles of all bilin lyases for phycoerythrin is not yet complete. To gain a more complete understanding about the individual functions of CpeZ and CpeY in PE biogenesis in cyanobacteria, we examined PE and phycobilisomes purified from wild type F. diplosiphon, cpeZ and cpeY knockout mutants. We find that the cpeZ and cpeY mutants accumulate less PE than wild type cells. We show that in the cpeZ mutant, chromophorylation of both PE subunits is affected, especially the Cys-80 and Cys-48/Cys-59 sites of CpeB, the beta-subunit of PE. The cpeY mutant showed reduced chromophorylation at Cys-82 of CpeA. We also show that, in vitro, CpeZ stabilizes PE subunits and assists in refolding of CpeB after denaturation. Taken together, we conclude that CpeZ acts as a chaperone-like protein, assisting in the folding/stability of PE subunits, allowing bilin lyases such as CpeY and CpeS to attach PEB to their PE subunit.

Mechanisms of L-triiodothyronine-induced inhibition of synaptosomal na(+)-k(+)-ATPase activity in young adult rat brain cerebral cortex.

The role of thyroid hormones (TH) in the normal functioning of adult mammalian brain is unclear. Our studies have identified synaptosomal Na(+)-K(+)-ATPase as a TH-responsive physiological parameter in adult rat cerebral cortex. L-triiodothyronine (T3) and L-thyroxine (T4) both inhibited Na(+)-K(+)-ATPase activity (but not Mg(2+)-ATPase activity) in similar dose-dependent fashions, while other metabolites of TH were less effective. Although both T3 and the ß -adrenergic agonist isoproterenol inhibited Na(+)-K(+)-ATPase activity in cerebrocortical synaptosomes in similar ways, the ß -adrenergic receptor blocker propranolol did not counteract the effect of T3. Instead, propranolol further inhibited Na(+)-K(+)-ATPase activity in a dose-dependent manner, suggesting that the effect of T3 on synaptosomal Na(+)-K(+)-ATPase activity was independent of ß -adrenergic receptor activation. The effect of T3 on synaptosomal Na(+)-K(+)-ATPase activity was inhibited by the α2-adrenergic agonist clonidine and by glutamate. Notably, both clonidine and glutamate activate Gi-proteins of the membrane second messenger system, suggesting a potential mechanism for the inhibition of the effects of TH. In this paper, we provide support for a nongenomic mechanism of action of TH in a neuronal membrane-related energy-linked process for signal transduction in the adult condition.

Structural and biochemical characterization of the bilin lyase CpcS from Thermosynechococcus elongatus.

Cyanobacterial phycobiliproteins have evolved to capture light energy over most of the visible spectrum due to their bilin chromophores, which are linear tetrapyrroles that have been covalently attached by enzymes called bilin lyases. We report here the crystal structure of a bilin lyase of the CpcS family from Thermosynechococcus elongatus (TeCpcS-III). TeCpcS-III is a 10-stranded ß barrel with two alpha helices and belongs to the lipocalin structural family. TeCpcS-III catalyzes both cognate as well as noncognate bilin attachment to a variety of phycobiliprotein subunits. TeCpcS-III ligates phycocyanobilin, phycoerythrobilin, and phytochromobilin to the alpha and beta subunits of allophycocyanin and to the beta subunit of phycocyanin at the Cys82-equivalent position in all cases. The active form of TeCpcS-III is a dimer, which is consistent with the structure observed in the crystal. With the use of the UnaG protein and its association with bilirubin as a guide, a model for the association between the native substrate, phycocyanobilin, and TeCpcS was produced.

Direct differentiation of A-ring single attachment versus A- and D-ring double attachment of phycoerythrobilin chromophores to phycobiliproteins using MALDI mass spectrometry.

Bilin chromophore attachment to phycobiliproteins is an enzyme-catalyzed post-translational modification process. Bilin-lyases attach a bilin chromophore to their cognate protein through a thioether bond between the chromophore and a cysteine moiety. Bilin chromophores are attached to their phycobiliproteins through the 3(1) carbon of the bilin. Double attachment may also occur, and in this case, carbons 3(1) and 18(1) of the bilin are both forming covalent linkages to cysteine moieties. There is a mass spectrometric limitation when examining tryptic peptides containing two (or more) cysteines if one seeks to ascertain whether chromopeptides are singly or doubly attached. The problem is that singly and doubly attached chromopeptides appear at the same m/z value; thus, up until the present, only NMR analysis has been successful at determining whether the chromophore is singly or doubly attached. We report in this work a new, fast and accurate method for discriminating singly from doubly attached chromophores using MALDI-TOF mass spectrometry. This method was developed from mass spectral analysis of chromopeptides that had undergone in vitro or in vivo attachment of bilin chromophores to phycobiliproteins. Distinction is based on a characteristic neutral loss that appears in the MALDI-TOF mass spectrum only when the bilin is singly attached.

Phycoerythrin-specific bilin lyase-isomerase controls blue-green chromatic acclimation in marine Synechococcus.

The marine cyanobacterium Synechococcus is the second most abundant phytoplanktonic organism in the world's oceans. The ubiquity of this genus is in large part due to its use of a diverse set of photosynthetic light-harvesting pigments called phycobiliproteins, which allow it to efficiently exploit a wide range of light colors. Here we uncover a pivotal molecular mechanism underpinning a widespread response among marine Synechococcus cells known as "type IV chromatic acclimation" (CA4). During this process, the pigmentation of the two main phycobiliproteins of this organism, phycoerythrins I and II, is reversibly modified to match changes in the ambient light color so as to maximize photon capture for photosynthesis. CA4 involves the replacement of three molecules of the green light-absorbing chromophore phycoerythrobilin with an equivalent number of the blue light-absorbing chromophore phycourobilin when cells are shifted from green to blue light, and the reverse after a shift from blue to green light. We have identified and characterized MpeZ, an enzyme critical for CA4 in marine Synechococcus. MpeZ attaches phycoerythrobilin to cysteine-83 of the α-subunit of phycoerythrin II and isomerizes it to phycourobilin. mpeZ RNA is six times more abundant in blue light, suggesting that its proper regulation is critical for CA4. Furthermore, mpeZ mutants fail to normally acclimate in blue light. These findings provide insights into the molecular mechanisms controlling an ecologically important photosynthetic process and identify a unique class of phycoerythrin lyase/isomerases, which will further expand the already widespread use of phycoerythrin in biotechnology and cell biology applications.

Characterization of the activities of the CpeY, CpeZ, and CpeS bilin lyases in phycoerythrin biosynthesis in Fremyella diplosiphon strain UTEX 481.

When grown in green light, Fremyella diplosiphon strain UTEX 481 produces the red-colored protein phycoerythrin (PE) to maximize photosynthetic light harvesting. PE is composed of two subunits, CpeA and CpeB, which carry two and three phycoerythrobilin (PEB) chromophores, respectively, that are attached to specific Cys residues via thioether linkages. Specific bilin lyases are hypothesized to catalyze each PEB ligation. Using a heterologous, coexpression system in Escherichia coli, the PEB ligation activities of putative lyase subunits CpeY, CpeZ, and CpeS were tested on the CpeA and CpeB subunits from F. diplosiphon. Purified His(6)-tagged CpeA, obtained by coexpressing cpeA, cpeYZ, and the genes for PEB synthesis, had absorbance and fluorescence emission maxima at 566 and 574 nm, respectively. CpeY alone, but not CpeZ, could ligate PEB to CpeA, but the yield of CpeA-PEB was lower than achieved with CpeY and CpeZ together. Studies with site-specific variants of CpeA(C82S and C139S), together with mass spectrometric analysis of trypsin-digested CpeA-PEB, revealed that CpeY/CpeZ attached PEB at Cys(82) of CpeA. The CpeS bilin lyase ligated PEB at both Cys(82) and Cys(139) of CpeA but very inefficiently; the yield of PEB ligated at Cys(82) was much lower than observed with CpeY or CpeY/CpeZ. However, CpeS efficiently attached PEB to Cys(80) of CpeB but neither CpeY, CpeZ, nor CpeY/CpeZ could ligate PEB to CpeB.

Attachment of noncognate chromophores to CpcA of Synechocystis sp. PCC 6803 and Synechococcus sp. PCC 7002 by heterologous expression in Escherichia coli.

Many cyanobacteria use brilliantly pigmented, multisubunit macromolecular structures known as phycobilisomes as antenna to enhance light harvesting for photosynthesis. Recent studies have defined the enzymes that synthesize phycobilin chromophores as well as many of the phycobilin lyase enzymes that attach these chromophores to their cognate apoproteins. The ability of the phycocyanin α-subunit (CpcA) to bind alternative linear tetrapyrrole chromophores was examined through the use of a heterologous expression system in Escherichia coli. E. coli strains produced phycocyanobilin, phytochromobilin, or phycoerythrobilin when they expressed 3Z-phycocyanobilin:ferredoxin oxidoreductase (PcyA), 3Z-phytochromobilin:ferredoxin oxidoreductase (HY2) from Arabidopsis thaliana, or phycoerythrobilin synthase (PebS) from the myovirus P-SSM4, respectively. CpcA from Synechocystis sp. PCC 6803 or Synechococcus sp. PCC 7002 was coexpressed in these strains with the phycocyanin α-subunit phycocyanobilin lyase, CpcE/CpcF, or the phycoerythrocyanin α-subunit phycocyanobilin isomerizing lyase, PecE/PecF, from Noctoc sp. PCC 7120. Both lyases were capable of attaching three different linear tetrapyrrole chromophores to CpcA; thus, up to six different CpcA variants, each with a unique chromophore, could be produced with this system. One of these chromophores, denoted phytoviolobilin, has not yet been observed naturally. The recombinant proteins had unexpected and potentially useful properties, which included very high fluorescence quantum yields and photochemical activity. Chimeric lyases PecE/CpcF and CpcE/PecF were used to show that the isomerizing activity that converts phycocyanobilin to phycoviolobilin resides with PecF and not PecE. Finally, spectroscopic properties of recombinant phycocyanin R-PCIII, in which the CpcA subunits carry a phycoerythrobilin chromophore, are described.

Effects of modified Phycobilin biosynthesis in the Cyanobacterium Synechococcus sp. Strain PCC 7002.

The pathway for phycocyanobilin biosynthesis in Synechococcus sp. strain PCC 7002 comprises two enzymes: heme oxygenase and phycocyanobilin synthase (PcyA). The phycobilin content of cells can be modified by overexpressing genes encoding alternative enzymes for biliverdin reduction. Overexpression of the pebAB and HY2 genes, encoding alternative ferredoxin-dependent biliverdin reductases, caused unique effects due to the overproduction of phycoerythrobilin and phytochromobilin, respectively. Colonies overexpressing pebAB became reddish brown and visually resembled strains that naturally produce phycoerythrin. This was almost exclusively due to the replacement of phycocyanobilin by phycoerythrobilin on the phycocyanin α-subunit. This phenotype was unstable, and such strains rapidly reverted to the wild-type appearance, presumably due to strong selective pressure to inactivate pebAB expression. Overproduction of phytochromobilin, synthesized by the Arabidopsis thaliana HY2 product, was tolerated much better. Cells overexpressing HY2 were only slightly less pigmented and blue-green than the wild type. Although the pcyA gene could not be inactivated in the wild type, pcyA was easily inactivated when cells expressed HY2. These results indicate that phytochromobilin can functionally substitute for phycocyanobilin in Synechococcus sp. strain PCC 7002. Although functional phycobilisomes were assembled in this strain, the overall phycobiliprotein content of cells was lower, the efficiency of energy transfer by these phycobilisomes was lower than for wild-type phycobilisomes, and the absorption cross-section of the cells was reduced relative to that of the wild type because of an increased spectral overlap of the modified phycobiliproteins with chlorophyll a. As a result, the strain producing phycobiliproteins carrying phytochromobilin grew much more slowly at low light intensity.

Phycobiliprotein biosynthesis in cyanobacteria: structure and function of enzymes involved in post-translational modification.

Cyanobacterial phycobiliproteins are brilliantly colored due to the presence of covalently attached chromophores called bilins, linear tetrapyrroles derived from heme. For most phycobiliproteins, these post-translational modifications are catalyzed by enzymes called bilin lyases; these enzymes ensure that the appropriate bilins are attached to the correct cysteine residues with the proper stereochemistry on each phycobiliprotein subunit. Phycobiliproteins also contain a unique, post-translational modification, the methylation of a conserved asparagine (Asn) present at beta-72, which occurs on the beta-subunits of all phycobiliproteins. We have identified and characterized several new families of bilin lyases, which are responsible for attaching PCB to phycobiliproteins as well as the Asn methyl transferase for beta-subunits in Synechococcus sp. PCC 7002 and Synechocystis sp. PCC 6803. All of the enzymes responsible for synthesis of holo-phycobiliproteins are now known for this cyanobacterium, and a brief discussion of each enzyme family and its role in the biosynthesis of phycobiliproteins is presented here. In addition, the first structure of a bilin lyase has recently been solved (PDB ID: 3BDR). This structure shows that the bilin lyases are most similar to the lipocalin protein structural family, which also includes the bilin-binding protein found in some butterflies.

Biosynthesis of cyanobacterial phycobiliproteins in Escherichia coli: chromophorylation efficiency and specificity of all bilin lyases from Synechococcus sp. strain PCC 7002.

Phycobiliproteins are water-soluble, light-harvesting proteins that are highly fluorescent due to linear tetrapyrrole chromophores, which makes them valuable as probes. Enzymes called bilin lyases usually attach these bilin chromophores to specific cysteine residues within the alpha and beta subunits via thioether linkages. A multiplasmid coexpression system was used to recreate the biosynthetic pathway for phycobiliproteins from the cyanobacterium Synechococcus sp. strain PCC 7002 in Escherichia coli. This system efficiently produced chromophorylated allophycocyanin (ApcA/ApcB) and alpha-phycocyanin with holoprotein yields ranging from 3 to 12 mg liter(-1) of culture. This heterologous expression system was used to demonstrate that the CpcS-I and CpcU proteins are both required to attach phycocyanobilin (PCB) to allophycocyanin subunits ApcD (alpha(AP-B)) and ApcF (beta(18)). The N-terminal, allophycocyanin-like domain of ApcE (L(CM)(99)) was produced in soluble form and was shown to have intrinsic bilin lyase activity. Lastly, this in vivo system was used to evaluate the efficiency of the bilin lyases for production of beta-phycocyanin.