The expression of a candidate cucumber fruit sugar starvation marker gene <Emphasis Type="Italic">CsSEF1</Emphasis> is enhanced in malformed fruit induced by salinity

The cucumber (Cucumis sativus L.) gene Cucumis sativus Somatic Embryogenesis Zinc Finger 1 (CsSEF1) was suggested to be a good marker gene for sugar starvation in fruit. The expression of this gene in fruits is dramatically upregulated in plants that have suffered either complete defoliation or prolonged darkness. CsSEF1 was initially discovered as a gene that was upregulated during somatic embryogenesis. We examined the difference in fruit parts and the effect of pollination on the upregulation of CsSEF1 induced by defoliation treatment. The results indicated that the upregulation of CsSEF1 in fruit by defoliation is not dependent on the presence of developing embryos. The expression of CsSEF1 was upregulated in malformed fruit induced by salinity in which the development of placenta was arrested. Partial cutting of the distal part of the fruit showed that if placenta tissue remained there was no upregulation of CsSEF1, whereas when placenta tissue did not remain there was a marked upregulation of CsSEF1. These results could be consistently interpreted as showing that placenta tissue induced the transport of photoassimilates to the fruit and that without developing placenta tissue, pericarp tissue suffers from severe sugar starvation. This interpretation, in turn, enforces the view that CsSEF1 is a good marker gene of fruit sugar starvation.     

Contrasted responses to carbohydrate limitation in tomato fruit at two stages of development

The metabolic consequences of long‐term carbohydrate depletion have been well documented in many sink organs but not extensively in fruit. Therefore, in the present study the response to sugar limitation in tomato fruit (Lycopersicon esculentum Mill.) was investigated at two developmental stages; during the cell division and cell expansion phases. First, the response in excised fruit cultured in vitro was characterized. Sugar depletion caused an arrest of growth and an exhaustion of carbon reserves. The proteins that were degraded and the nitrogen released was transiently stored as asparagine and glutamine in both developmental stages and also as γ ‐aminobutyric acid (GABA) in expanding fruit. Fruit at the cell division stage appeared to be more sensitive to sugar limitation. The response to sugar depletion was then characterized in fruit from plants submitted to extended darkness. In planta, the effects of sugar‐limitation were similar to those described in vitro but much more attenuated, especially in expanding fruit, which still accumulated dry matter. The expression of cell cycle genes, sugar‐ and nitrogen‐related genes was reduced by darkness. Only asparagine synthetase gene expression was induced in both dark‐treated fruit. Together the present data revealed that the effects of the carbon limitation are more pronounced in the youngest fruits as it is probably controlled by the relative sink strength of the fruit.     

HECATE2 acts with GLABROUS3 and Tu to boost cytokinin biosynthesis and regulate cucumber fruit wart formation

Warty fruit in cucumber (Cucumis sativus L.) is an important quality trait that greatly affects fruit appearance and market value. The cucumber wart consists of fruit trichomes (spines) and underlying tubercules, in which the existence of spines is prerequisite for tubercule formation. Although several regulators have been reported to mediate spine or tubercule formation, the direct link between spine and tubercule development remains unknown. Here, we found that the basic Helix-Loop-Helix (bHLH) gene HECATE2 (CsHEC2) was highly expressed in cucumber fruit peels including spines and tubercules. Knockout of CsHEC2 by the CRISPR/Cas9 system resulted in reduced wart density and decreased cytokinin (CTK) accumulation in the fruit peel, whereas overexpression of CsHEC2 led to elevated wart density and CTK level. CsHEC2 is directly bound to the promoter of the CTK hydroxylase-like1 gene (CsCHL1) that catalyzes CTK biosynthesis, and activated CsCHL1 expression. Moreover, CsHEC2 physically interacted with GLABROUS3 (CsGL3, a key spine regulator) and Tuberculate fruit (CsTu, a core tubercule formation factor), and such interactions further enhanced CsHEC2-mediated CsCHL1 expression. These data suggested that CsHEC2 promotes wart formation by acting as an important cofactor for CsGL3 and CsTu to directly stimulate CTK biosynthesis in cucumber. Thus, CsHEC2 can serve as a valuable target for molecular breeding of cucumber varieties with different wart density requirements.

Fruit wart is a key external quality trait, and CsHECATE2 promotes wart formation by interacting with a spine regulator and a tubercule factor to directly stimulate cytokinin biosynthesis in cucumber     

Nitrogen Assimilating Enzyme Activities and Enzyme Protein during Development and Senescence of Effective and Plant Gene-Controlled Ineffective Alfalfa Nodules

Effective (N₂-fixing) alfalfa (Medicago sativa L.) and plant-controlled ineffective (non-N₂-fixing) alfalfa recessive for the in₁ gene were compared to determine the effects of the in₁ gene on nodule development, acetylene reduction activity (ARA), and nodule enzymes associated with N assimilation and disease resistance. Effective nodule ARA reached a maximum before activities of glutamine synthetase (GS), glutamate synthase (GOGAT), aspartate aminotransferase (AAT), asparagine synthetase (AS), and phosphoenolpyruvate carboxylase (PEPC) peaked. Ineffective nodule ARA was only 5% of effective nodule ARA. Developmental profiles of GS, GOGAT, AAT, and PEPC activities were similar for effective and ineffective nodules, but activities in ineffective nodules were lower and declined earlier. Little AS activity was detected in developing ineffective nodules. Changes in GS, GOGAT, AAT, and PEPC activities in developing and senescent effective and ineffective nodules generally paralleled amounts of immunologically detectable enzyme polypeptides. Effective nodule GS, GOGAT, AAT, AS, and PEPC activities declined after defoliation. Activities of glutamate dehydrogenase, malate dehydrogenase, phenylalanine ammonia lyase, and caffeic acid-o-methyltransferase were unrelated to nodule effectiveness. Maximum expression of nodule N-assimilating enzymes appeared to require the continued presence of a product associated with effective bacteroids that was lacking in in₁ effective nodules.     

Asparagine synthesis in pea leaves, and the occurrence of an asparagine synthetase inhibitor

Asparagine is present in the mature leaves of young pea (Pisum sativum cv Little Marvel) seedlings, and is synthesized in detached shoots. This accumulation and synthesis is greatly enhanced by darkening. In detached control shoots, [¹⁴C]aspartate was metabolized predominantly to organic acids and, as other workers have shown, there was little labeling of asparagine (after 5 hours, 3.1% of metabolized label). Addition of the aminotransferase inhibitor aminooxyacetate decreased the flow of aspartate carbon to organic acids and enhanced (about 3-fold) the labeling of asparagine. The same treatment applied to darkened shoots resulted in a substantial conversion of [¹⁴C]aspartate to asparagine, over 10-fold greater than in control shoots (66% of metabolized label), suggesting that aspartate is the normal precursor of asparagine.

Only traces of glutamine-dependent asparagine synthetase activity could be detected in pea leaf or root extracts; activity was not enhanced by sulfhydryl reagents, oxidizing conditions, or protease inhibitors. Asparagine synthetase is readily extracted from lupin cotyledons, but yield was greatly reduced by extraction in the presence of pea leaf tissue; pea leaf homogenates contained an inhibitor which produced over 95% inhibition of an asparagine synthetase preparation from lupin cotyledons. The inhibitor was heat stable, with a low molecular weight. Presence of an inhibitor may prevent detection of asparagine synthetase in pea extracts and in Asparagus, where a cyanide-dependent pathway has been proposed to account for asparagine synthesis: an inhibitor with similar properties was present in Asparagus shoot tissue.

     

Identification and Functional Characterization of a Novel Bacterial Type Asparagine Synthetase A: A tRNA SYNTHETASE PARALOG FROM LEISHMANIA DONOVANI*

Asparagine is formed by two structurally distinct asparagine synthetases in prokaryotes. One is the ammonia-utilizing asparagine synthetase A (AsnA), and the other is asparagine synthetase B (AsnB) that uses glutamine or ammonia as a nitrogen source. In a previous investigation using sequence-based analysis, we had shown that Leishmania spp. possess asparagine-tRNA synthetase paralog asparagine synthetase A (LdASNA) that is ammonia-dependent. Here, we report the cloning, expression, and kinetic analysis of ASNA from Leishmania donovani. Interestingly, LdASNA was both ammonia- and glutamine-dependent. To study the physiological role of ASNA in Leishmania, gene deletion mutations were attempted via targeted gene replacement. Gene deletion of LdASNA showed a growth delay in mutants. However, chromosomal null mutants of LdASNA could not be obtained as the double transfectant mutants showed aneuploidy. These data suggest that LdASNA is essential for survival of the Leishmania parasite. LdASNA enzyme was recalcitrant toward crystallization so we instead crystallized and solved the atomic structure of its close homolog from Trypanosoma brucei (TbASNA) at 2.2 Å. A very significant conservation in active site residues is observed between TbASNA and Escherichia coli AsnA. It is evident that the absence of an LdASNA homolog from humans and its essentiality for the parasites make LdASNA a novel drug target.     

Reassimilation of Photorespiratory Ammonium in Lotus japonicus Plants Deficient in Plastidic Glutamine Synthetase

It is well established that the plastidic isoform of glutamine synthetase (GS₂) is the enzyme in charge of photorespiratory ammonium reassimilation in plants. The metabolic events associated to photorespiratory NH₄⁺ accumulation were analyzed in a Lotus japonicus photorespiratory mutant lacking GS₂. The mutant plants accumulated high levels of NH₄⁺ when photorespiration was active, followed by a sudden drop in the levels of this compound. In this paper it was examined the possible existence of enzymatic pathways alternative to GS₂ that could account for this decline in the photorespiratory ammonium. Induction of genes encoding for cytosolic glutamine synthetase (GS₁), glutamate dehydrogenase (GDH) and asparagine synthetase (ASN) was observed in the mutant in correspondence with the diminishment of NH₄⁺. Measurements of gene expression, polypeptide levels, enzyme activity and metabolite levels were carried out in leaf samples from WT and mutant plants after different periods of time under active photorespiratory conditions. In the case of asparagine synthetase it was not possible to determine enzyme activity and polypeptide content; however, an increased asparagine content in parallel with the induction of ASN gene expression was detected in the mutant plants. This increase in asparagine levels took place concomitantly with an increase in glutamine due to the induction of cytosolic GS₁ in the mutant, thus revealing a major role of cytosolic GS₁ in the reassimilation and detoxification of photorespiratory NH₄⁺ when the plastidic GS₂ isoform is lacking. Moreover, a diminishment in glutamate levels was observed, that may be explained by the induction of NAD(H)-dependent GDH activity.     

A genetic map of melon highly enriched with fruit quality QTLs and EST markers, including sugar and carotenoid metabolism genes

A genetic map of melon enriched for fruit traits was constructed, using a recombinant inbred (RI) population developed from a cross between representatives of the two subspecies of Cucumis melo L.: PI 414723 (subspecies agrestis) and ‘Dulce’ (subspecies melo). Phenotyping of 99 RI lines was conducted over three seasons in two locations in Israel and the US. The map includes 668 DNA markers (386 SSRs, 76 SNPs, six INDELs and 200 AFLPs), of which 160 were newly developed from fruit ESTs. These ESTs include candidate genes encoding for enzymes of sugar and carotenoid metabolic pathways that were cloned from melon cDNA or identified through mining of the International Cucurbit Genomics Initiative database (http://www.icugi.org/). The map covers 1,222 cM with an average of 2.672 cM between markers. In addition, a skeleton physical map was initiated and 29 melon BACs harboring fruit ESTs were localized to the 12 linkage groups of the map. Altogether, 44 fruit QTLs were identified: 25 confirming QTLs described using other populations and 19 newly described QTLs. The map includes QTLs for fruit sugar content, particularly sucrose, the major sugar affecting sweetness in melon fruit. Six QTLs interacting in an additive manner account for nearly all the difference in sugar content between the two genotypes. Three QTLs for fruit flesh color and carotenoid content were identified. Interestingly, no clear colocalization of QTLs for either sugar or carotenoid content was observed with over 40 genes encoding for enzymes involved in their metabolism. The RI population described here provides a useful resource for further genomics and metabolomics studies in melon, as well as useful markers for breeding for fruit quality.     

Relaxed tRNA specificity of the Staphylococcus aureus aspartyl-tRNA synthetase enables RNA-dependent asparagine biosynthesis

The human pathogen Staphylococcus aureus is an asparagine prototroph despite its genome not encoding an asparagine synthetase. S. aureus does use an asparaginyl-tRNA synthetase (AsnRS) to directly ligate asparagine to tRNA^Asn. The S. aureus genome also codes for one aspartyl-tRNA synthetase (AspRS). Here we demonstrate the lone S. aureus aspartyl-tRNA synthetase has relaxed tRNA specificity and can be used with the amidotransferase GatCAB to synthesize asparagine on tRNA^Asn. S. aureus thus encodes both the direct and indirect routes for Asn-tRNA^Asn formation while encoding only one aspartyl-tRNA synthetase. The presence of the indirect pathway explains how S. aureus synthesizes asparagine without either asparagine synthetase.     

Identification and Expression Analysis of D-type Cyclin Genes in Early Developing Fruit of Cucumber (Cucumis sativus L.)

Plant D-type cyclin genes (CYCDs) are important regulators of cell division. However, little is known on their participation during the early developmental stage of cucumber fruit. In this study, cucumber CYCD genes were identified and characterized. The expression levels of these genes during early fruit development were assessed from 0 to 8 days after anthesis (DAA). The results revealed the presence of 13 different CYCD genes, which were named according to identity percentages of the corresponding orthologs in Arabidopsis thaliana and poplar. The genomic organization of each subgroup CYCD was similar to their orthologs in A. thaliana and poplar. The expression levels of CsCYCD genes were analyzed in cucumber fruits under different treatments including natural parthenocarpic fruit, pollinated fruit, and N-(2-chloro-4-pyidyl)-N′-phenyurea (CPPU)-induced parthenocarpic fruit. The highest expression levels of most CsCYCDs genes were at four DAA in natural parthenocarpic and pollinated fruits. Interestingly, the expression patterns of 8 of 13 CsCYCD genes in natural parthenocarpic fruit were similar to those in pollinated fruit, but different from those in CPPU-induced parthenocarpic fruit. Collectively, the results of this study provide insights on the CYCDs involved in cucumber parthenocarpic fruit development.     

Inheritance and mapping of the ore gene controlling the quantity of β-carotene in cucumber (Cucumis sativus L.) endocarp

The metabolic precursor of vitamin A, ??-carotene, is essential for human health. The gene(s) controlling ??-carotene quantity (Q??C) has been introgressed from Xishuangbanna gourd (XIS, possessing ??-carotene; Cucumis sativus L. var. xishuangbannanesis Qi et Yuan; 2n?=?2x?=?14) into cultivated cucumber (no ??-carotene; Cucumis sativus L.). To determine the inheritance of Q??C in cucumber fruit endocarp, F1 progeny and a set of 124 F7 recombinant inbred lines (RILs) derived from the cultivated cucumber line CC3 and XIS line SWCC8 were evaluated for Q??C during 2009 and 2010 in Nanjing, China. Segregation analysis revealed that endocarp Q??C of greenhouse-grown fruit was controlled by a single recessive gene. Further, marker analysis indicated the gene controlling Q??C was linked to seven SSR markers on linkage group 3, where their order was SSR20710?CSSR19511?CSSR15419?CSSR07706?Core?CSSR23231?CSSR11633?CSSR20270. These markers and the putative candidate gene were mapped to cucumber chromosome 3DS. An evaluation of 30 genetically diverse cucumber lines indicated that marker SSR07706 has utility in further genetic analyses of the Q??C orange endocarp gene, designated ore. Moreover, the markers defined herein may have utility for marker-assisted selection directed towards the development of cucumber germplasm with high fruit ??-carotene content.     

Asparagine synthetases of Klebsiella aerogenes: properties and regulation of synthesis

We isolated pleiotropic mutants of Klebsiella aerogenes with the transposon Tn5 which were unable to utilize a variety of poor sources of nitrogen. The mutation responsible was shown to be in the asnB gene, one of two genes coding for an asparagine synthetase. Mutations in both asnA and asnB were necessary to produce an asparagine requirement. Assays which could distinguish the two asparagine synthetase activities were developed in strains missing a high-affinity asparaginase. The asnA and asnB genes coded for ammonia-dependent and glutamine-dependent asparagine synthetases, respectively. Asparagine repressed both enzymes. When growth was nitrogen limited, the level of the ammonia-dependent enzyme was low and that of the glutamine-dependent enzyme was high. The reverse was true in a nitrogen-rich (ammonia-containing) medium. Furthermore, mutations in the glnG protein, a regulatory component of the nitrogen assimilatory system, increased the level of the ammonia-dependent enzyme. The glutamine-dependent asparagine synthetase was purified to 95%. It was a tetramer with four equal 57,000-dalton subunits and catalyzed the stoichiometric generation of asparagine, AMP, and inorganic pyrophosphate from aspartate, ATP, and glutamine. High levels of ammonium chloride (50 mM) could replace glutamine. The purified enzyme exhibited a substrate-independent glutaminase activity which was probably an artifact of purification. The tetramer could be dissociated; the monomer possessed the high ammonia-dependent activity and the glutaminase activity, but not the glutamine-dependent activity. In contrast, the purified ammonia-dependent asparagine synthetase, about 40% pure, had a molecular weight of 80,000 and is probably a dimer of identical subunits. Asparagine inhibited both enzymes. Kinetic constants and the effect of pH, substrate, and product analogs were determined. The regulation and biochemistry of the asparagine synthetases prove the hypothesis strongly suggested by the genetic and physiological evidence that a glutamine-dependent enzyme is essential for asparagine synthesis when the nitrogen source is growth rate limiting.