Molecular characterisation of apple accessions with respect to aminocyclopropane-1-carboxylic acid synthase gene (ACS1) polymorphism

CITATION: Mhelembe K. et al. 2020. Molecular characterisation of apple accessions with respect to aminocyclopropane-1-carboxylic acid synthase gene (ACS1) polymorphism. Horticultural Science (Prague), 47:69-79. doi:10.17221/83/2018-HORTSCIThe original publication is available at https://www.agriculturejournals.cz/web/hortsci/The ARC apple gene bank collection was genotyped for the fruit expressed gene ACS1, in which a short-interspersed element (SINE) in the promoter is known, when homozygous, to correlate with the delayed ethylene production. Primers were designed amplifying products less than 500 bp and 224 cultivars of domestic apple were analysed, 169 not previously genotyped. Of these, 82 were aa (homozygous for the high ethylene allele at 202 bp), 73 were ab and 14 bb (homozygous for the low ethylene allele, with the SINE, at 339 bp). The difference between the allele sizes, 137 bp, observed in the current study is consistent with the indel of 138 bp originally described, but differs considerably from the indel of 166 bp reported in literature. In addition, 21 accessions of other Malus species were analysed. Only one, M. ‘Golden Hornet’, had the b allele, which suggests it may have been introgressed from M. pumila.https://www.agriculturejournals.cz/web/hortsci.htm?type=article&id=83_2018-HORTSCIPublishers versio

Glucose-induced posttranslational activation of protein phosphatases PP2A and PP1 in yeast

The protein phosphatases PP2A and PP1 are major regulators of a variety of cellular processes in yeast and other eukaryotes. Here, we reveal that both enzymes are direct targets of glucose sensing. Addition of glucose to glucose-deprived yeast cells triggered rapid posttranslational activation of both PP2A and PP1. Glucose activation of PP2A is controlled by regulatory subunits Rts1, Cdc55, Rrd1 and Rrd2. It is associated with rapid carboxymethylation of the catalytic subunits, which is necessary but not sufficient for activation. Glucose activation of PP1 was fully dependent on regulatory subunits Reg1 and Shp1. Absence of Gac1, Glc8, Reg2 or Red1 partially reduced activation while Pig1 and Pig2 inhibited activation. Full activation of PP2A and PP1 was also dependent on subunits classically considered to belong to the other phosphatase. PP2A activation was dependent on PP1 subunits Reg1 and Shp1 while PP1 activation was dependent on PP2A subunit Rts1. Rts1 interacted with both Pph21 and Glc7 under different conditions and these interactions were Reg1 dependent. Reg1-Glc7 interaction is responsible for PP1 involvement in the main glucose repression pathway and we show that deletion of Shp1 also causes strong derepression of the invertase gene SUC2. Deletion of the PP2A subunits Pph21 and Pph22, Rrd1 and Rrd2, specifically enhanced the derepression level of SUC2, indicating that PP2A counteracts SUC2 derepression. Interestingly, the effect of the regulatory subunit Rts1 was consistent with its role as a subunit of both PP2A and PP1, affecting derepression and repression of SUC2, respectively. We also show that abolished phosphatase activation, except by reg1Δ, does not completely block Snf1 dephosphorylation after addition of glucose. Finally, we show that glucose activation of the cAMP-PKA (protein kinase A) pathway is required for glucose activation of both PP2A and PP1. Our results provide novel insight into the complex regulatory role of these two major protein phosphatases in glucose regulation

Signaling mechanisms for nutrient activation of protein kinases and phosphatases in yeast.

For microorganisms, nutrients represent one of the key environmental determinants controlling many aspects of cell function, including growth and proliferation. For example, addition of amino acids to nitrogen-deprived, glucose-grown cells of the yeast Saccharomyces cerevisiae triggers a rapid reversal of the stationary phase phenotype that characterizes such cells, i.e. trehalose and glycogen are mobilized, and cells lose their overall stress resistance and resume growth. Two major nutrient‑sensing pathways in yeast, the cAMP-Protein Kinase A (PKA) pathwayand the Fermentable Growth Medium-induced (FGM) pathway, couple nutrient cues to the cell s physiological and developmental program. Whereas the cAMP-PKA pathway is activated in response to fermentable carbon sources, activation of the FGM pathway requires a complete growth medium in which nitrogen, sulphur, phosphate and a fermentable carbon source are present in adequate amounts. In addition to its well-documented function as an amino acid transporter, it has recently been shown that the general amino acid permease Gap1 also acts as a receptor. The dual function of Gap1 as amino acid transporter/receptor (transceptor) was supported by the isolation of constitutively activating alleles. These Gap1 mutant alleles contain short truncations, e.g. 14 amino acids for Gap1DC6(14aa), of the extreme carboxyl tail of the permease, with their expression resulting in a constitutively high PKA phenotype. This was observed under all growth conditions tested,even in medium with ammonium as sole nitrogen source. In the first part of this study, we focused on characterizing the high PKA phenotype observed in cells carrying these truncated Gap1 mutant proteins. By blocking known nutrient signaling pathways, either through thedisruption of an activating protein or the use of specific inhibitors, we show that the phenomenon is not caused by overactivation of either the rapamycin-sensitive TORC1 kinase, the phosphoinositide 4-kinase Stt4 or the protein kinase Sch9. We also provide experimental evidence that the mutant proteins mediate the overactive PKA phenotype in a cyclic AMP (cAMP)‑independent, but PKA-dependent manner. Subsequent work showed that the overactive signaling phenotype is synergistically dependent on both the truncated permease and a background mutation in the specific S1278b gap1 D strain used, called seg1-1 . The seg1-1 mutation, for Suppressor of ER exit-deficient Gap1 , causes the secretionto the plasma membrane of truncated Gap1 alleles that lack a C‑terminally‑based ER exit signal which, in wild-type cells, are retained in the ER by the organelle s quality control system. Genetic evidence indicated that the seg1-1 mutation is both single and recessive. We employed a novel genetic mapping technology developed in our laboratory, AMTEM, for identification of the wild-type counterpart of seg1-1 . AMTEM makes use of artificially‑marked yeast strains which contain 600 different markers, inserted at neutral positions throughout the whole genome. In the screening process, we identified a 40 kb region between bp 18,226 and 58,935 near the beginning of chromosome VIII that is linked to the high PKA phenotype. Based on our earlierdata, we first focused on a candidate gene (within the determined region) which functions within the yeast s secretion pathway, namely the Golgi-localized v-SNARE GOS1 . Through detailed biochemical analyses, we showed that the expression of Gap1DC6(14aa) in gos1 D cells causes PKA phenotypes identical to those observed in seg1-1 Gap1DC6(14aa) cells, i.e. trehalose levels remain very low, even when these mutantsare starved for nitrogen. Sequence analysis of the GOS1 ORF, promoter and terminator regions in the seg1-1 background, however, didnot reveal any missense or other potentially relevant mutations. We sequenced the rest of the 40 kb region, and identified a point mutation within the ECM29 ORF, located at bp 5524. ECM29 is the next gene on chromosome VIII, but located on the opposite strand to GOS1 . The nucleotide at bp 5524, an adenine in both the S288c and the S1278b wild-type strains, is changed to a guanine in the seg1-1 mutant. This leads to an amino acid change in which the amino acid residue at position 1842, an asparagine (N), is substituted for aspartic acid (D). The role that Ecm29 may play in the overactive signaling phenotype isnot clear yet, and requires further investigation. The next part of this study deals with the mechanism by which glucose addition to glucose-deprived yeast cells causes rapid activation of the serine/threonine protein phosphatases PP2A and PP1. Previous work has shown that both PP2A and PP1 are under direct control of glucose sensing. Here, we convincingly demonstrate that glucose-induced activation of PP2Arequires the catalytic subunits Pph21 and Pph22. Of the PP2A regulatorysubunits, only Rts1 is involved; Cdc55 appears to be dispensable for this activity. Furthermore, we show that cells deficient in catalytic subunit methylation exhibit a slight decrease in PP2A activation upon glucose addition. Moreover, methylation of PP2A s catalytic subunits increasesin response to glucose re-addition. We also identified four different regulatory subunits required for glucose-induced activation of PP1, namely Shp1, Glc8, Gac1 and Red1. Strainslacking Gac1 or Red1 show only a partial decrease in glucose-induced activation of PP1, compared to the shp1 D and glc8 D mutants that exhibit a complete loss of PP1-specific activation. We attribute the partial decrease in PP1 activation observed in gac1 D and red1 D cells to an overlap in function.status: publishe

Development of synthetic signal sequences for heterologous protein secretion from Saccharomyces cerevisiae

Thesis (MSc)--Stellenbosch University, 2003.ENGLISH ABSTRACT: Protein secretion and intracellular transport are highly regulated processes and involve the interplay of a multitude of proteins. A unique collection of thermosensitive secretory mutants allowed scientists to demonstrate that the secretory pathway of the yeast Saccharomyces cerevisiae is very similar to that of the higher eukaryotes. All proteins commence their journey in the endoplasmic reticulum, where they undergo amino-linked core glycosyl modification. After passage through the Golgi apparatus, where the remodelling of the glycosyl chains is completed, proteins are transported to their final destinations, which are either the cell surface, periplasmic space or the vacuole. Proteins destined for secretion are usually synthesised with a transient amino-terminal secretion leader of varying length and hydrophobicity, which plays a crucial role in the targeting and translocation of their protein cargo. Considerable effort has been made to elucidate the molecular mechanisms involved in these processes, especially due to their relevance in a rapidly expanding biotech industry. The advantages of S. cerevisiae as a host for the expression of recombinant proteins are well documented. Unfortunately, S. cerevisiae is also subject to a number of drawbacks, with a relative low product yield being one of the major disadvantages. Bearing this in mind, different secretion leaders were compared with the aim of improving the secretion of the LKA 1 and LKA2 a-amylase enzymes from the S. cerevisiae secretion system. The yeast Lipomyces kononenkoae is well known for its ability to degrade raw starch and an improved secretion of its amylase enzymes from S. cerevisiae paves the way for a potential one-step starch utilisation process. Three sets of constructs were prepared containing the LKA 1 and LKA2 genes separately under secretory direction of either their native secretion leader, the S. cerevisiae mating pheromone a-factor (MFa1) secretion leader, or the MFa1 secretion leader containing a synthetic C-terminal spacer peptide (EEGEPK). The inclusion of a spacer peptide in the latter set of constructs ensured improved Kex2p proteolytic processing of the leader/protein fusion. Strains expressing the amylase genes under their native secretion leaders resulted in the highest saccharolytic activity in the culture medium. In contrast to this, strains utilising the synthetic secretion leader produced the highest fermentation yield, but had a lower than expected extracellular activity. We hypothesise that the native amylase leaders may function as intramolecular chaperones in the folding and processing of their passenger proteins, thereby increasing processing efficiency and concomitant enzyme activity.AFRIKAANSE OPSOMMING: Proteïensekresie en intrasellulêre transport is hoogs gereguleerde prosesse en betrek die onderlinge wisselwerking van 'n verskeidenheid proteïene. 'n Unieke versameling van temperatuur-sensitiewe sekresiemutante het wetenskaplikes in staat gestelom die ooreenkoms tussen die sekresiepad van die gis Saccharomyces cerevisiae en dié van komplekser eukariote aan te toon. Alle proteïene begin hul reis in die endoplasmiese retikulum, waartydens hulle ook amino-gekoppelde kernglikosielveranderings ondergaan. Nadat die proteïene deur die Golgi-apparaat beweeg het, waar die laaste veranderings aan die glikosielkettings plaasvind, word hulle na hul finale bestemmings, waaronder die seloppervlak, die periplasmiese ruimte of die vakuool, vervoer. Proteïene wat vir sekresie bestem is, word gewoonlik met 'n tydelike, amino-eindpuntsekresiesein, wat 'n kritiese rol in die teiken en translokasie van hul proteïenvrag speel, gesintetiseer. Heelwat pogings is in hierdie studie aangewend om die molekulêre meganismes betrokke by hierdie prosesse te ontrafel, veral as gevolg van hul toepaslikheid in 'n vinnig groeiende biotegnologiebedryf. Die voordele van S. cerevisiae as 'n gasheer vir die uitdruk van rekombinante proteïene is alombekend. S. cerevisiae het egter ook verskeie nadele, waaronder die relatiewe lae produkopbrengs die belangrikste is. Teen hierdie agtergrond, is verskillende sekresieseine met mekaar vergelyk met die doelom die sekresie van die LKA 1 en LKA2 a-amilasegene vanuit die S. cerevisiae-uitdrukkingsisteem te verbeter. Die gis Lipomyces kononenkoae is bekend vir sy vermoeë om rou stysel af te breek en 'n verbeterde sekresie van sy amilasegene vanuit S. cerevisiae baan die weg vir 'n moontlike een-stap styselgebruiksproses. Drie stelle konstrukte is gemaak wat die LKA 1- en LKA2- gene onafhanklik onder sekresiebeheer van onderskeidelik hul inheemse sekresiesein, die S. cerevisiae paringsferomoonsekresiesein (MFa1) of die MFa1-sekresiesein met 'n sintetiese koppelingspeptied aan die C-eindpunt (EEGEPK), plaas. Die insluiting van 'n koppelingspeptied in die laasgenoemde stel konstrukte verseker verbeterde Kex2p proteolitiese prosessering van die sein/proteïenfusie. Rasse wat die amilasegene onder beheer van hul inheemse sekresieseine uitdruk, het die beste saccharolitiese aktiwiteit in die kultuurmedia getoon. In teenstelling hiermee, het rasse wat van die sintetiese sekresiesein gebruik maak, die beste fermentasie-opbrengs getoon, maar met 'n laer as verwagte ekstrasellulêre aktiwiteit. Ons vermoed dat die inheemse amilaseseine as intramolekulêre begeleiers optree in die vou en prosessering van hul proteïenpassasiers, wat lei tot verbeterde prosessering en ensiemaktiwiteit

Applying volumetric electron microscopy to visualize xylem tissue impacted by citrus tristeza virus-induced stem pitting

Citrus tristeza virus (CTV) causes several disease syndromes in different citrus hosts; namely quick decline, seedling yellows and stem pitting. CTV-induced stem pitting leads to substantial economic losses in sensitive citrus varieties, including grapefruit. The formation of stem pits has previously been linked to the ability of the virus to colonize xylem tissue outside of its typical phloem limitation, thereby disrupting normal xylem development. The nature of this compromised tissue has not been fully elucidated. In this study, stem pits were characterized at the molecular anatomical level using a combination of techniques to better understand the characteristics of the xylem and phloem tissues impacted by severe pitting. Biological staining was used to visualize CTV-induced stem pitting and was complemented with a novel technology that has not previously been used to study CTV-induced stem pitting, namely serial block-face scanning electron microscopy (SBF-SEM). This proof-of-concept study yielded new insights into the morphology of stem pitting-affected tissue. The utility of SBF-SEM for stem pitting characterization was also demonstrated and an optimized protocol for its application on hard, woody material is presented

Biodistribution of a potential chemotherapeutic, dinuclearbisphosphinogold(I) dithiocarbamate, as determined by its 198Au radiolabelled analogue

Dinuclearbisphosphinogold(I) dithiocarbamato, BPDTC, was previously found to have antitumour activity in vitro. 198Au radiolabelled BPDTC (radiochemical yield of 70 ± 6 % and radiochemical purity of[95 %) was used to determine its in vivo biodistribution in Sprague-Dawley rats. Gamma scintigraphs were performed over a period of 48 h and final radioactivity measurements of harvested organs of the test animals after termination was performed at 2, 4 and 48 h. The study successfully showed the biodistribution of the gold complex, with the highest uptake of the compound being observed in the lungs, liver and spleenNuclear Technologies in Medicine and the Biosciences Initiative (NTeMBI), a national technology platform developed and managed by the South African Nuclear Energy Corporation (Necsa) and funded by the Department of Science and Technology. Biomed (Mintek

Control of Mould Spoilage on Apples Using Yeasts as Biological Control Agents

Considerable quantities of fruit are lost during pre- and post-harvest stages due to mould spoilage. The aim of this study was to evaluate the antagonistic effect of selected yeasts against spoilage mould Botrytis cinerea, Penicillium expansum and Alternaria alstroemeriae. One hundred and four yeast isolates were screened for antagonistic activity against B. cinerea, P. expansum and A. alstroemeriae using radial inhibition, dual and mouth-to-mouth plate assays. Sixty-seven out of 104 yeasts showed growth inhibition activity against P. expansum, while 36 yeasts inhibited B. cinerea, 47 yeasts inhibited A. alstroemeriae, but only 22 yeasts showed inhibition activity against all three moulds. Candida pyralidae Y63, Meyerozyma guilliermondii Y88 and Zygoascus hellenicus Y89 showed highest inhibition activity against all three moulds, when mode of inhibition was due to direct contact. Volatile organic compounds produced by Pichia kluyveri Y64, C. pyralidae Y63 and M. guilliermondii Y88 showed the highest growth inhibition against all three moulds. These yeasts were also evaluated against all three moulds on apples. P. kluyveri Y64 showed 100%, 57% and 26% growth inhibition against A. alstroemeriae, B. cinerea and P. expansum, respectively, on apples and performed slightly better than a commercial fungicide against B. cinerea and P. expansum. While M. guillermondii Y88 showed 100%, 60% and 18% inhibition on apples against A. alstroemeriae, B. cinerea and P. expansum, respectively. P. kluyveri Y64 and M. guilliermondii Y88 showed potential as biofungicides and warrant further investigation

Novel mechanisms in nutrient activation of the yeast Protein Kinase A pathway

In yeast the Protein Kinase A (PKA) pathway can be activated by a variety of nutrients. Fermentable sugars, like glucose and sucrose, trigger a spike in the cAMP level, followed by activation of PKA and phosphorylation of target proteins causing a.o. mobilization of reserve carbohydrates, repression of stress-related genes and induction of growth-related genes. Glucose and sucrose are sensed by a G-protein coupled receptor system that activates adenylate cyclase and also activates a bypass pathway causing direct activation of PKA. Addition of other essential nutrients, like nitrogen sources or phosphate, to glucose-repressed nitrogen-or phosphate-starved cells, also triggers rapid activation of the PKA pathway. In these cases cAMP is not involved as a second messenger. Amino acids are sensed by the Gap1 transceptor, previously considered only as an amino acid transporter. Recent results indicate that the amino acid ligand has to induce a specific conformational change for signaling. The same amino acid binding site is involved in transport and signaling. Similar results have been obtained for Pho84 which acts as a transceptor for phosphate activation of the PKA pathway. Ammonium activation of the PKA pathway in nitrogen-starved cells is mediated mainly by the Mep2 transceptor, which belongs to a different class of transporter proteins. Hence, different types of sensing systems are involved in control of the yeast PKA pathway by nutrients