Dihydrodipicolinate synthase is absent in fungi Desbois, Sebastien; John, Ulrik P.; Perugini, Matthew A.
Biochimie,
September 2018, 2018-Sep, 2018-09-00, 20180901, Volume:
152
Journal Article
Peer reviewed
The class I aldolase dihydrodipicolinate synthase (DHDPS) catalyzes the first committed step of the diaminopimelate (DAP) lysine biosynthesis pathway in bacteria, archaea and plants. Despite the ...existence, in databases, of numerous fungal sequences annotated as DHDPS, its presence in fungi has been the subject of contradictory claims. We report the characterization of DHDPS candidates from fungi. Firstly, the putative DHDPS from Coccidioides immitis (PDB ID: 3QFE) was shown to have negligible enzyme activity. Sequence analysis of 3QFE showed that three out of the seven amino acid residues critical for DHDPS activity are absent; however, exact matches to catalytic residues from two other class I aldolases, 2-keto-3-deoxygluconate aldolase (KDGA), and 4-hydroxy-2-oxoglutarate aldolase (HOGA), were identified. The presence of both KDGA and HOGA activity in 3QFE was confirmed in vitro using enzyme assays, the first report of such dual activity. Subsequent analyses of all publically available fungal sequences revealed that no entry contains all seven residues important for DHDPS function. The candidate with the highest number of identities (6 of 7), KIW77228 from Fonsecaea pedrosoi, was shown to have trace DHDPS activity in vitro, partially restored by substitution of the seventh critical residue, and to be incapable of complementing DHDPS-deficient E. coli cells. Combined with the presence of all seven sequences for the alternative α-aminoadipate (AAA) lysine biosynthesis pathway in C. immitis and F. pedrosoi, we believe that DHDPS and the DAP pathway are absent in fungi, and further, that robust informed methods for annotating genes need to be implemented.
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•Sequence databases contain numerous fungal sequences misannotated as DHDPS.•Annotated fungi DHDPS are other class I aldolases including a dual active HOGA/KDGA.•A 7 residue sequence motif is described for correctly annotating DHDPS sequences.•Bioinformatics analyses confirm that fungi use the AAA pathway to synthesize lysine.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK, ZRSKP
Summary
Cleistogenes songorica (2n = 4x = 40) is a desert grass with a unique dimorphic flowering mechanism and an ability to survive extreme drought. Little is known about the genetics underlying ...drought tolerance and its reproductive adaptability. Here, we sequenced and assembled a high‐quality chromosome‐level C. songorica genome (contig N50 = 21.28 Mb). Complete assemblies of all telomeres, and of ten chromosomes were derived. C. songorica underwent a recent tetraploidization (~19 million years ago) and four major chromosomal rearrangements. Expanded genes were significantly enriched in fatty acid elongation, phenylpropanoid biosynthesis, starch and sucrose metabolism, and circadian rhythm pathways. By comparative transcriptomic analysis we found that conserved drought tolerance related genes were expanded. Transcription of CsMYB genes was associated with differential development of chasmogamous and cleistogamous flowers, as well as drought tolerance. Furthermore, we found that regulation modules encompassing miRNA, transcription factors and target genes are involved in dimorphic flower development, validated by overexpression of CsAP2_9 and its targeted miR172 in rice. Our findings enable further understanding of the mechanisms of drought tolerance and flowering in C. songorica, and provide new insights into the adaptability of native grass species in evolution, along with potential resources for trait improvement in agronomically important species.
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BFBNIB, DOBA, FZAB, GIS, IJS, IZUM, KILJ, NLZOH, NUK, OILJ, PILJ, PNG, SAZU, SBCE, SBMB, UILJ, UKNU, UL, UM, UPUK
ABSTRACT
The cryophilic Antarctic hair grass, Deschampsia antarctica E. Desv., one of two higher plants indigenous to Antarctica, represents a unique resource for the study of freeze tolerance ...mechanisms. We have previously characterized a multi‐gene family in D. antarctica encoding ice recrystallization inhibition proteins (IRIPs) whose transcript levels are responsive to cold acclimation, and whose products confer ice recrystallization inhibition (RI) activity that can account for activity seen in cold acclimated plants. We used molecular and physiological analyses to investigate temporal responses of D. antarctica to cold acclimation and de‐acclimation, and sub‐zero acclimation. Quantitative profiling revealed that IRIP transcript levels significantly increased and decreased within hours of cold acclimation and de‐acclimation, respectively, becoming up to 1000‐fold more abundant in fully acclimated plants. Western analysis detected three major immuno‐reactive bands whose pattern of accumulation mirrored that of transcript. These data correlated with the onset and decline of RI activity in acclimated and de‐acclimated leaves. Plant survival‐based testing revealed that cold acclimation enhanced freeze tolerance by 5 °C within 4 d, and that sub‐zero acclimation conferred an additional 3 °C of tolerance. Thus, D. antarctica is highly responsive to temperature fluctuations, able to rapidly deploy IRIP based RI activity and enhance its freeze tolerance.
A comprehensive time course analysis of the cryophilic Antarctic hairgrass Deschampsia antarctica has revealed that it is highly responsive to temperature fluctuations and able to rapidly deploy freeze tolerance mechanisms. We have investigated the kinetics of accumulation of ice recrystallisation inhibition protein (IRIP) gene encoded transcript and protein products, and the recrystallisation inhibition (RI) activity that they confer, in response to cold and sub‐zero acclimation, and de‐acclimation. We have also correlated these findings with a novel, field relevant, measure of plant freeze tolerance. This work provides, insight into the mechanisms that D. antarctica uses to contend with such an extreme and variable environment and, further evidence of the role of IRIPs in freeze tolerance.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
Antarctic hair grass (Deschampsia antarctica E. Desv.), the only grass indigenous to Antarctica, has well-developed freezing tolerance, strongly induced by cold acclimation. Here, we show that in ...response to low temperatures, D. antarctica expresses potent recrystallization inhibition (RI) activity that, inhibits the growth of small ice crystals into potentially damaging large ones, is proteinaceous and localized to the apoplasm. A gene family from D. antarctica encoding putative homologs of an ice recrystallization inhibition protein (IRIP) has been isolated and characterized. IRIPs are apoplastically targeted proteins with two potential ice-binding motifs: 1-9 leucine-rich repeats (LRRs) and c. 16 'IRIP' repeats. IRIP genes appear to be confined to the grass subfamily Pooideae and their products, exhibit sequence similarity to phytosulphokine receptors and are predicted to adopt conformations with two ice-binding surfaces. D. antarctica IRIP (DaIRIP) transcript levels are greatly enhanced in leaf tissue following cold acclimation. Transgenic Arabidopsis thaliana expressing a DaIRIP has novel RI activity, and purified DaIRIP, when added back to extracts of leaves from non-acclimated D. antarctica, can reconstitute the activity found in acclimated plants. We propose that IRIP-mediated RI activity may contribute to the cryotolerance of D. antarctica, and thus to its unique ability to have colonized Antarctica.
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BFBNIB, FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
In the fission yeast Schizosaccharomyces pombe, the protein kinase Chk1 has an essential role in transducing a delay signal to the cell cycle machinery in the presence of DNA damage. Fission yeast ...cells lacking the chk1 gene do not delay progression of the cell cycle in response to damage and are thus sensitive to DNA damaging agents. We have previously shown that Chk1 is phosphorylated following DNA damage induced by a variety of agents and that this is dependent on the integrity of the DNA damage checkpoint pathway, including Rad3, the ATR homolog. Through a combination of mutagenesis and phospho-specific antibodies, we have shown that serine at position 345 (S345) is phosphorylated in vivo in response to DNA damage, and that S345 phosphorylation is required for an intact checkpoint response. We have developed a kinase assay for Chk1, and have shown that basal Chk1 kinase activity is increased in response to DNA damage and that this increase, but not the basal activity, is dependent on S345. Furthermore, we show that S345 phosphorylation is required for Chk1 to associate with Rad24, a 14-3-3 protein, upon DNA damage. These results are consistent with a model whereby Chk1 phosphorylation results in increased Chk1 kinase activity that is necessary for both checkpoint delay and cellular survival following damage to the genome. These data are similar to observations made in mammalian cells and Xenopus oocyte extracts, suggesting that mechanisms leading to Chk1 activation have been conserved in evolution.
► Our findings illustrate the potential for targeted functional genomic studies in non-model, non-crop plant species, particularly in extremophiles, to identify novel or enhanced stress tolerance ...mechanisms. ► A gene discovery and transcript abundance profiling study has been undertaken in
Cleistogenes songorica, a grass that thrives in severely water depleted environments in Inner Mongolia. ► Amongst an EST collection from drought stressed plants are many sequences with roles in response to drought stress. ► Transcript abundance profiling revealed that the majority of genes tested were regulated by drought. ► These findings offer a functional insight into the molecular mechanisms used by
C. songorica to cope with moisture limitation, and identify potential resources for conferring drought tolerance in sensitive plants.
Cleistogenes songorica is an important perennial grass found in the pastoral steppe of Inner Mongolia.
C. songorica flourishes in drought prone environments, and therefore provides an ideal candidate plant system for the identification of drought-tolerance conferring genes. We constructed cDNA libraries from leaves and roots of drought-stressed
C. songorica seedlings. Expressed sequence tag (EST) sequencing of 5664 random cDNA clones produced 3579 high quality, trimmed sequences. The average read length of trimmed ESTs was 613
bp. Clustering and assembly identified a non-redundant set of 1499 contigs, including 805 singleton unigenes and 694 multi-member unigenes. The resulting unigenes were functionally categorized according to the Gene Ontology (GO) hierarchy using the in house Bioinformatic Advanced Scientific Computing (BASC) annotation pipeline. Among the total 2.2
Mbp of EST sequence data, 161 putative SSRs were found, a frequency similar to that previously observed in oat and
Arabidopsis ESTs. Sixty-three unigenes were functionally annotated as being stress responsive, of which 22 were similar to genes implicated in drought stress response. Using quantitative real time RT-PCR, transcripts of 13 of these 22 genes were shown to be at least three fold more, or less abundant in drought-stressed leaves or roots, with 8 increased and 5 decreased in relative transcript abundance. The
C. songorica EST and cDNA collections generated in this study are a valuable resource for microarray-based expression profiling, and functional genomics in order to elucidate their role, and to understand the underlying mechanisms of drought-tolerance in
C. songorica.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPUK
Pin1/Ess1p is a highly conserved WW domain-containing peptidyl-prolyl isomerase (PPIase); its WW domain binds specifically to phospho-Ser/Thr-Pro sequences and its catalytic domain isomerizes ...phospho-Ser/Thr-Pro bonds. Pin1 PPIase activity can alter protein conformation in a phosphorylation-dependent manner and/or promote protein dephosphorylation. Human Pin1 interacts with mitotic phosphoproteins, such as NIMA, Cdc25 and Wee1, and inhibits G(2)/M progression in Xenopus extracts. Depletion of Pin1 in HeLa cells and deletion of ESS1 in S. cerevisiae result in mitotic arrest. In addition, Pin1/Ess1p play roles in transcription in S. cerevisiae and in mammalian somatic cells. The S. pombe genome sequence has an open reading frame (ORF) that has 47% identity with Pin1. Expression of this ORF rescued the growth defect caused by ess1 deletion in S. cerevisiae, indicating that S. pombe Pin1p is a functional Pin1 homologue. Overexpression of pin1(+) in S. pombe caused slow growth and a G(1) delay. Deletion of pin1(+) (pin1 Delta) did not affect cell cycle progression or cell growth, but increased sensitivity to the cyclophilin inhibitor, cyclosporin A, suggesting that cyclophilin family PPIases have overlapping functions with the Pin1p PPIase. Deletion of pin1(+) did not affect the DNA replication checkpoint, but conferred a modest increase in UV sensitivity. Furthermore, the pin1 Delta allele caused a synthetic growth defect when combined with either cdc25-22 or wee1-50 but not the cdc24-1 temperature-sensitive mutant. The pin1 Delta strain showed increased sensitivity to the PP1/PP2A family phosphatase inhibitor, okadaic acid, suggesting that Pin1p plays a role in protein dephosphorylation as a result of its ability to increase the population of phospho-Ser/Thr-Pro peptide bonds in the trans conformation that is required for PP2A-mediated dephosphorylation. Our genetic data also suggest that Pin1p might function as a positive regulator of Cdc25p and Wee1p.
NIMA kinases appear to be the least functionally conserved mitotic regulators, being implicated in chromosome condensation in fungi and in spindle function in metazoans. We demonstrate here that the ...fission yeast NIMA homologue, Fin1p, can induce profound chromosome condensation in the absence of the condensin and topoisomerase II, indicating that Fin1p‐induced condensation differs from mitotic condensation. Fin1p expression is transcriptionally and post‐translationally cell cycle‐regulated, with Fin1p kinase activity maximal from the metaphase–anaphase transition to G1. Fin1p is localized to the spindle pole body and fin1Δ cells are hypersensitive to anti‐microtubule drugs, synthetically lethal with a number of spindle mutants and require the spindle checkpoint for viability. Moreover, fin1Δ cells show unusual and extensive elaborations of the nuclear envelope. These data support a role for Fin1p in spindle function and nuclear envelope transactions at or after the metaphase–anaphase transition that may be generally applicable to other NIMA‐family members.
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FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SBCE, SBMB, UL, UM, UPUK
Supernumerary B chromosomes represent one of many causes of numerical chromosome variation that exist in higher plants and animals. Sequences of DNA unique to B chromosomes of Brachycome ...dichromosomatica were enriched prior to cloning and resultant clones hybridizing only to plants containing B chromosomes were further investigated. Sequences of DNA that were characterised include members of a family of 176-bp tandem repeats that are specific to the B chromosomes of B. dichromosomatica, an annual Australian native plant species with only two pairs of A chromosomes and up to three dispensable B chromosomes. Sequence analysis of these six related clones indicated that some regions of the sequence are more highly conserved than others or, alternatively, that some adenine residues at the NdeII site are methylated. The repeat is homologous to DNA from Brachycome ciliaris var. languinosa but not to DNA from other related taxa growing in the vicinity of the B. dichromosomatica populations.
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