Ups and downs of a transcriptional landscape shape iron deficiency associated chlorosis of the maize inbreds B73 and Mo17

BACKGROUND: Improving nutrient homeostasis is a major challenge of a sustainable maize cultivation, and cornerstone to ensure food supply for a growing world population. Although, iron constitutes an important nutrient, iron availability is limited. In this respect, iron deficiency associated chlorosis causes severe yield losses every year. Natural variation of the latter trait has yet not been addressed in maize and was therefore studied in the present analysis. RESULTS: In this study, we i) report about the contrasting chlorosis phenotypes of the inbreds B73 and Mo17 at 10 and 300 μM iron regime, ii) identified over 400 significantly regulated transcripts (FDR < 0.05) within both inbreds at these growth conditions by deep RNA-Sequencing, iii) linked the gained knowledge with QTL information about iron deficiency related traits within the maize intermated B73 by Mo17 (IBM) population, and iv) highlighted contributing molecular pathways. In this respect, several genes within methionine salvage pathway and phytosiderophore synthesis were found to present constitutively high expression in Mo17, even under sufficient iron supply. Moreover, the same expression pattern could be observed for two putative bHLH transcription factors. In addition, a number of differentially expressed genes showed a co-localisation with QTL confidence intervals for iron deficiency related traits within the IBM population. CONCLUSIONS: Our study highlights differential iron deficiency associated chlorosis between B73 and Mo17 and represents a valuable resource for differentially expressed genes upon iron limitation and chlorosis response. Besides identifying two putative bHLH transcription factors, we propose that methionine salvage pathway and sterol metabolism amongst others; underlie the contrasting iron deficiency related chlorosis phenotype of both inbreds. Altogether, this study emphasizes a contribution of selected genes and pathways on natural trait variation within the IBM population

Association genetics in Solanum tuberosum provides new insights into potato tuber bruising and enzymatic tissue discoloration

<p>Abstract</p> <p>Background</p> <p>Most agronomic plant traits result from complex molecular networks involving multiple genes and from environmental factors. One such trait is the enzymatic discoloration of fruit and tuber tissues initiated by mechanical impact (bruising). Tuber susceptibility to bruising is a complex trait of the cultivated potato (<it>Solanum tuberosum</it>) that is crucial for crop quality. As phenotypic evaluation of bruising is cumbersome, the application of diagnostic molecular markers would empower the selection of low bruising potato varieties. The genetic factors and molecular networks underlying enzymatic tissue discoloration are sparsely known. Hitherto there is no association study dealing with tuber bruising and diagnostic markers for enzymatic discoloration are rare.</p> <p>Results</p> <p>The natural genetic diversity for bruising susceptibility was evaluated in elite middle European potato germplasm in order to elucidate its molecular basis. Association genetics using a candidate gene approach identified allelic variants in genes that function in tuber bruising and enzymatic browning. Two hundred and five tetraploid potato varieties and breeding clones related by descent were evaluated for two years in six environments for tuber bruising susceptibility, specific gravity, yield, shape and plant maturity. Correlations were found between different traits. In total 362 polymorphic DNA fragments, derived from 33 candidate genes and 29 SSR loci, were scored in the population and tested for association with the traits using a mixed model approach, which takes into account population structure and kinship. Twenty one highly significant (p < 0.001) and robust marker-trait associations were identified.</p> <p>Conclusions</p> <p>The observed trait correlations and associated marker fragments provide new insight in the molecular basis of bruising susceptibility and its natural variation. The markers diagnostic for increased or decreased bruising susceptibility will facilitate the combination of superior alleles in breeding programs. In addition, this study presents novel candidates that might control enzymatic tissue discoloration and tuber bruising. Their validation and characterization will increase the knowledge about the underlying biological processes.</p

Charakterisierung und Bedeutung von ausgewählten Transportproteinen für die Virulenz von phytopathogenen Bakterien und Pilzen

Durch vorangegangene Arbeiten wurden transgene Kartoffelpflanzen erzeugt, die eine verringerte Aktivität des plastidären ATP/ADP Transporters aufweisen (NTT-antisense). Die Knollen dieser Pflanzen zeigen nicht nur veränderte Gehalte an Primärmetaboliten sondern auch eine erhöhte Pathogen-Resistenz, zum Beispiel gegen den bakteriellen Krankheitserreger Erwinia carotovora ssp. atroseptica (Eca). In diesem Zusammenhang deuten neuere Veröffentlichungen auf eine Bedeutung von Transportproteinen für Wirt-Pathogen Wechselwirkungen hin. Im Zuge dieser Arbeit konnte durch die Herstellung eines Systems zur gezielten Erzeugung von Eca „K.O.“-Mutanten die Bedeutung von ausgewählten Sequenzen, welche für Transportproteine kodieren, analysiert werden. Hierbei zeigte sich, dass im Bezug auf die Virulenz von Eca, sowohl der Prolin- als auch der d-Galaktonattransport nur von untergeordneter Bedeutung ist. In weiteren Untersuchungen wurde allerdings der Karbonsäuremetabolismus und -transport von Eca als zentrales Element in der Entfaltung maximaler bakterieller Virulenz erkannt. Hierbei wurde das Cit1-Protein als hoch-affiner Citrattransporter, welcher über die bakterielle pmf energetisiert wird, identifiziert. Dieses Protein ist für ein Wachstum von Eca auf Citrat als einziger Kohlenstoffquelle notwendig und essentiell zur Etablierung einer vollständigen Pathogenese auf Kartoffelknollenscheibchen. So weisen Eca cit1 „K.O.“-Mutanten im Vergleich zu Wildtyp Zellen nicht nur eine geringere pektolytische Aktivität und Gewebemazeration, sondern auch ein reduziertes in planta Wachstum, auf. Des Weiteren wurde ermittelt, dass sich NTT-antisense Gewebe nicht nur durch eine erhöhte Pathogen-Resistenz auszeichnet, sondern auch erniedrigte Citratgehalte aufweist. Analog führte eine artifizielle Erhöhung des Citratgehaltes durch Infiltration von Knollengewebe zu einer deutlich erhöhten Gewebemazeration durch Eca sowie einer verringerten Akkumulation von Abwehrrelevanten pflanzlichen Gentranskripten (PR-Gene). Weitere untersuchte Eca-Mutanten belegten, dass im gemeinsamen Wirken mit anderen enzymatischen Komponenten, Citrat ein ambivalentes Molekül für pflanzliche Resistenz und bakterielle Virulenz ist. Weiterhin konnte gezeigt werden, dass Transportproteine nicht nur für die Virulenz von Eca, sondern auch für phytopathogene Pilze wie Magnaporthe grisea, von Bedeutung sind. So konnte durch die Kooperation mit der AG Thines (IBWF) das Genprodukt eines bei Pathogenese induzierten Genes (rig2), biochemisch charakterisiert werden. Hierbei wurde mittels Komplementation einer Hefemutante (22&#8710;8AA) nachgewiesen, dass es sich bei diesem Protein um einen Prolin-Transporter handelt. Diese Arbeit zeigt, dass Transportproteine als ein wichtiges Element in Wirt-Pathogen Beziehungen wirken können und eine Charakterisierung solcher Proteine zum Verständnis dieser Wechselwirkungen unerlässlich ist.Transgenic potato tubers exhibiting reduced levels of the plastidic ATP/ADP transporter (NTT antisense) show altered primary metabolite levels and substantially increased resistance against the necrotrophic plant pathogen Erwinia carotovora ssp. atroseptica (Eca). Investigations by several research groups on erwinial gene transcription identified transportproteins as putative virulence factors. Analysis of specific Eca carriers by means of targeted mutagenesis relying on a newly developed knock-out system (this work) led to the conclusion that both proline and d-galactonate uptake are not essential for erwinial virulence on potato tuber discs. In similiar experiments Eca virulence seemed to depend on a functional carboxylate metabolism and uptake. Further investigations showed that citrate uptake plays a crucial role for Eca in establishing tissue maceration. To analyse the effect of citrate import into Eca cells on the bacterial virulence we studied the function of various carboxylate-carrier proteins. One of the corresponding, ECA3984, encodes a highly specific citrate transporter (EcaCit1) energised by the proton-motive force. Knock-out mutants lacking the functional EcaCit1 protein did not grow in medium containing citrate as the sole carbon source and showed a substantially reduced ability to macerate potato tuber tissue and a reduced in-planta growth. Furthermore we discovered that NTT-antisense tubers do not only display a enhanced pathogen resistance but also markedly decreased levels of citrate. Citrate feeding into both, Eca resistant (NTT-antisense) and wild type tuber tissue resulted in an increased maceration efficiency. In addition citrate feeding into wild type and NTT-antisense tuber tissue decreased the expression of pathogen related genes upon challenging the tuber tissue with elicitors of plant resistance. Therefore citrate uptake into Eca is critical for full bacterial virulence and low citrate levels in potato tuber tissue contribute to the resistance against this pathogen. The impact of transportproteins on virulence is not restricted to Eca but was also demonstrated for Magnaporthe grisea, the rice blast fungus. Specific genes of M. grisea induced upon pathogenesis and coding for transporters were characterised on the molecular and phytopathogenic level by the research group Thines (IBWF). In this work the biochemical characterisation of one of these carriers was achieved. By complementation studies on yeast mutants the Rig2 of M. grisea protein was identified to be a proline-carrier. The results of this work show that some transportproteins can be considered as virulence factors and that the characterisation of such proteins allows a deeper understanding of host-pathogen interactions and the involved key-metabolites

Charakterisierung und Bedeutung von ausgewählten Transportproteinen für die Virulenz von phytopathogenen Bakterien und Pilzen

Durch vorangegangene Arbeiten wurden transgene Kartoffelpflanzen erzeugt, die eine verringerte Aktivität des plastidären ATP/ADP Transporters aufweisen (NTT-antisense). Die Knollen dieser Pflanzen zeigen nicht nur veränderte Gehalte an Primärmetaboliten sondern auch eine erhöhte Pathogen-Resistenz, zum Beispiel gegen den bakteriellen Krankheitserreger Erwinia carotovora ssp. atroseptica (Eca). In diesem Zusammenhang deuten neuere Veröffentlichungen auf eine Bedeutung von Transportproteinen für Wirt-Pathogen Wechselwirkungen hin. Im Zuge dieser Arbeit konnte durch die Herstellung eines Systems zur gezielten Erzeugung von Eca „K.O.“-Mutanten die Bedeutung von ausgewählten Sequenzen, welche für Transportproteine kodieren, analysiert werden. Hierbei zeigte sich, dass im Bezug auf die Virulenz von Eca, sowohl der Prolin- als auch der d-Galaktonattransport nur von untergeordneter Bedeutung ist. In weiteren Untersuchungen wurde allerdings der Karbonsäuremetabolismus und -transport von Eca als zentrales Element in der Entfaltung maximaler bakterieller Virulenz erkannt. Hierbei wurde das Cit1-Protein als hoch-affiner Citrattransporter, welcher über die bakterielle pmf energetisiert wird, identifiziert. Dieses Protein ist für ein Wachstum von Eca auf Citrat als einziger Kohlenstoffquelle notwendig und essentiell zur Etablierung einer vollständigen Pathogenese auf Kartoffelknollenscheibchen. So weisen Eca cit1 „K.O.“-Mutanten im Vergleich zu Wildtyp Zellen nicht nur eine geringere pektolytische Aktivität und Gewebemazeration, sondern auch ein reduziertes in planta Wachstum, auf. Des Weiteren wurde ermittelt, dass sich NTT-antisense Gewebe nicht nur durch eine erhöhte Pathogen-Resistenz auszeichnet, sondern auch erniedrigte Citratgehalte aufweist. Analog führte eine artifizielle Erhöhung des Citratgehaltes durch Infiltration von Knollengewebe zu einer deutlich erhöhten Gewebemazeration durch Eca sowie einer verringerten Akkumulation von Abwehrrelevanten pflanzlichen Gentranskripten (PR-Gene). Weitere untersuchte Eca-Mutanten belegten, dass im gemeinsamen Wirken mit anderen enzymatischen Komponenten, Citrat ein ambivalentes Molekül für pflanzliche Resistenz und bakterielle Virulenz ist. Weiterhin konnte gezeigt werden, dass Transportproteine nicht nur für die Virulenz von Eca, sondern auch für phytopathogene Pilze wie Magnaporthe grisea, von Bedeutung sind. So konnte durch die Kooperation mit der AG Thines (IBWF) das Genprodukt eines bei Pathogenese induzierten Genes (rig2), biochemisch charakterisiert werden. Hierbei wurde mittels Komplementation einer Hefemutante (22&#8710;8AA) nachgewiesen, dass es sich bei diesem Protein um einen Prolin-Transporter handelt. Diese Arbeit zeigt, dass Transportproteine als ein wichtiges Element in Wirt-Pathogen Beziehungen wirken können und eine Charakterisierung solcher Proteine zum Verständnis dieser Wechselwirkungen unerlässlich ist.Transgenic potato tubers exhibiting reduced levels of the plastidic ATP/ADP transporter (NTT antisense) show altered primary metabolite levels and substantially increased resistance against the necrotrophic plant pathogen Erwinia carotovora ssp. atroseptica (Eca). Investigations by several research groups on erwinial gene transcription identified transportproteins as putative virulence factors. Analysis of specific Eca carriers by means of targeted mutagenesis relying on a newly developed knock-out system (this work) led to the conclusion that both proline and d-galactonate uptake are not essential for erwinial virulence on potato tuber discs. In similiar experiments Eca virulence seemed to depend on a functional carboxylate metabolism and uptake. Further investigations showed that citrate uptake plays a crucial role for Eca in establishing tissue maceration. To analyse the effect of citrate import into Eca cells on the bacterial virulence we studied the function of various carboxylate-carrier proteins. One of the corresponding, ECA3984, encodes a highly specific citrate transporter (EcaCit1) energised by the proton-motive force. Knock-out mutants lacking the functional EcaCit1 protein did not grow in medium containing citrate as the sole carbon source and showed a substantially reduced ability to macerate potato tuber tissue and a reduced in-planta growth. Furthermore we discovered that NTT-antisense tubers do not only display a enhanced pathogen resistance but also markedly decreased levels of citrate. Citrate feeding into both, Eca resistant (NTT-antisense) and wild type tuber tissue resulted in an increased maceration efficiency. In addition citrate feeding into wild type and NTT-antisense tuber tissue decreased the expression of pathogen related genes upon challenging the tuber tissue with elicitors of plant resistance. Therefore citrate uptake into Eca is critical for full bacterial virulence and low citrate levels in potato tuber tissue contribute to the resistance against this pathogen. The impact of transportproteins on virulence is not restricted to Eca but was also demonstrated for Magnaporthe grisea, the rice blast fungus. Specific genes of M. grisea induced upon pathogenesis and coding for transporters were characterised on the molecular and phytopathogenic level by the research group Thines (IBWF). In this work the biochemical characterisation of one of these carriers was achieved. By complementation studies on yeast mutants the Rig2 of M. grisea protein was identified to be a proline-carrier. The results of this work show that some transportproteins can be considered as virulence factors and that the characterisation of such proteins allows a deeper understanding of host-pathogen interactions and the involved key-metabolites

QTL mapping identifies novel major loci for kernel row number-associated ear fasciation, ear prolificacy and tillering in maize (Zea mays L.)

Maize ear fasciation originates from excessive or abnormal proliferation of the ear meristem and usually manifests as flattened multiple-tipped ear and/or disordered kernel arrangement. Ear prolificacy expresses as multiple ears per plant or per node. Both ear fasciation and prolificacy can affect grain yield. The genetic control of the two traits was studied using two recombinant inbred line populations (B73 x Lo1016 and Lo964 x Lo1016) with Lo1016 and Lo964 as donors of ear fasciation and prolificacy, respectively. Ear fasciation-related traits, number of kernel rows (KRN), ear prolificacy and number of tillers were phenotyped in multi-year field experiments. Ear fasciation traits and KRN showed relatively high heritability (h(2) &gt; 0.5) except ratio of ear diameters. For all ear fasciation-related traits, fasciation level positively correlated with KRN (0.30 &lt;= r &lt;= 0.68). Prolificacy and tillering were not correlated and their h(2) ranged from 0.41 to 0.78. QTL mapping identified four QTLs for ear fasciation, on chromosomes 1 (two QTLs), 5 and 7, the latter two overlapping with QTLs for number of kernel rows. Notably, at these QTLs, the Lo1016 alleles increased both ear fasciation and KRN across populations, thus showing potential breeding applicability. Four and five non-overlapping QTLs were mapped for ear prolificacy and tillering, respectively. Two ear fasciation QTLs, qFas1.2 and qFas7, overlapped with fasciation QTLs mapped in other studies and spanned compact plant2 and ramosa1 candidate genes. Our study identified novel ear fasciation loci and alleles positively affecting grain yield components, and ear prolificacy and tillering loci which are unexpectedly still segregating in elite maize materials, contributing useful information for genomics-assisted breeding programs

The Nucleotide Transporter of Caedibacter caryophilus Exhibits an Extended Substrate Spectrum Compared to the Analogous ATP/ADP Translocase of Rickettsia prowazekii

The two obligate intracellular alphaproteobacteria Rickettsia prowazekii and Caedibacter caryophilus, a human pathogen and a paramecium endosymbiont, respectively, possess transport systems to facilitate ATP uptake from the host cell cytosol. These transport proteins, which have 65% identity at the amino acid level, were heterologously expressed in Escherichia coli, and their properties were compared. The results presented here demonstrate that the caedibacter transporter had a broader substrate than the more selective rickettsial transporter. ATP analogs with modified sugar moieties, dATP and ddATP, inhibited the transport of ATP by the caedibacter transporter but not by the rickettsial transporter. Both transporters were specific for di- and trinucleotides with an adenine base in that adenosine tetraphosphate, AMP, UTP, CTP, and GTP were not competitive inhibitors. Furthermore, the antiporter nature of both transport systems was shown by the dependence of the efflux of [α-(32)P]ATP on the influx of substrate (ATP but not dATP for rickettsiae, ATP or dATP for caedibacter)