Multi-Omics of Tomato Glandular Trichomes Reveals Distinct Features of Central Carbon Metabolism Supporting High Productivity of Specialized Metabolites

Glandular trichomes are metabolic cell factories with the capacity to produce large quantities of secondary metabolites. Little is known about the connection between central carbon metabolism and metabolic productivity for secondary metabolites in glandular trichomes. To address this gap in our knowledge, we performed comparative metabolomics, transcriptomics, proteomics, and (13)C-labeling of type VI glandular trichomes and leaves from a cultivated (Solanum lycopersicum LA4024) and a wild (Solanum habrochaites LA1777) tomato accession. Specific features of glandular trichomes that drive the formation of secondary metabolites could be identified. Tomato type VI trichomes are photosynthetic but acquire their carbon essentially from leaf sucrose. The energy and reducing power from photosynthesis are used to support the biosynthesis of secondary metabolites, while the comparatively reduced Calvin-Benson-Bassham cycle activity may be involved in recycling metabolic CO2 Glandular trichomes cope with oxidative stress by producing high levels of polyunsaturated fatty acids, oxylipins, and glutathione. Finally, distinct mechanisms are present in glandular trichomes to increase the supply of precursors for the isoprenoid pathways. Particularly, the citrate-malate shuttle supplies cytosolic acetyl-CoA and plastidic glycolysis and malic enzyme support the formation of plastidic pyruvate. A model is proposed on how glandular trichomes achieve high metabolic productivity

Combined 15N-Labeling and TandemMOAC Quantifies Phosphorylation of MAP Kinase Substrates Downstream of MKK7 in Arabidopsis

Reversible protein phosphorylation is a widespread posttranslational modification that plays a key role in eukaryotic signal transduction. Due to the dynamics of protein abundance, low stoichiometry and transient nature of protein phosphorylation, the detection and accurate quantification of substrate phosphorylation by protein kinases remains a challenge in phosphoproteome research. Here, we combine tandem metal-oxide affinity chromatography (tandemMOAC) with stable isotope 15N metabolic labeling for the measurement and accurate quantification of low abundant, transiently phosphorylated peptides by mass spectrometry. Since tandemMOAC is not biased toward the enrichment of acidophilic, basophilic, or proline-directed kinase substrates, the method is applicable to identify targets of all these three types of protein kinases. The MKK7-MPK3/6 module, for example, is involved in the regulation of plant development and plant basal and systemic immune responses, but little is known about downstream cascade components. Using our here described phosphoproteomics approach we identified several MPK substrates downstream of the MKK7-MPK3/6 phosphorylation cascade in Arabidopsis. The identification and validation of dynamin-related protein 2 as a novel phosphorylation substrate of the MKK7-MPK3/6 module establishes a novel link between MPK signaling and clathrin-mediated vesicle trafficking

Genetic structure of Hypochaeris uniflora (Asteraceae) suggests vicariance in the Carpathians and rapid post-glacial colonization of the Alps from an eastern Alpine refugium

International audienceAim The range of the subalpine species Hypochaeris uniflora covers the Alps, Carpathians and Sudetes Mountains. Whilst the genetic structure and post-glacial history of many high-mountain plant taxa of the Alps is relatively well documented, the Carpathian populations have often been neglected in phylogeographical studies. The aim of the present study is to compare the genetic variation of the species in two major European mountain systems - the Alps and the Carpathians. Locations Alps and Carpathians. Methods The genetic variation of 77 populations, each consisting of three plants, was studied using amplified fragment length polymorphism (AFLP). Results Neighbour joining and principal coordinate analyses revealed three well-supported phylogeographical groups of populations corresponding to three disjunct geographical regions - the Alps and the western and south-eastern Carpathians. Moreover, two further clusters could be distinguished within the latter mountain range, one consisting of populations from the eastern Carpathians and the second consisting of populations from the southern Carpathians. Populations from the Apuseni Mountains had an intermediate position between the eastern and southern Carpathians. The genetic clustering of populations into four groups was also supported by an analysis of molecular variance, which showed that most genetic variation (almost 46%) was found among these four groups. By far the highest within-population variation was found in the eastern Carpathians, followed by populations from the southern and western Carpathians. Generally, the populations from the Alps were considerably less variable and displayed substantially fewer region-diagnostic markers than those from the south-eastern Carpathians. Although no clear geographical structure was found within the Alps, based on neighbour joining or principal coordinate analyses, some trends were obvious: populations from the easternmost part were genetically more variable and, together with those from the south-western part, exhibited a higher proportion of rare AFLP fragments than populations in other areas. Moreover, the total number of AFLP fragments per population, the percentage of polymorphic loci and the proportion of rare AFLP fragments significantly decreased from east to west. Main conclusions Deep infraspecific phylogeographical gaps between the populations from the Alps and the western and south-eastern Carpathians suggest the survival of H. uniflora in three separate refugia during the last glaciation. Our AFLP data provide molecular evidence for a long-term geographical disjunction between the eastern and western Carpathians, previously suggested from the floristic composition at the end of 19th century. It is likely that Alpine populations survived the Last Glacial in the eastern part of the Alps, from where they rapidly colonized the rest of the Alps after the ice sheet retreated. Multiple founder effects may explain a gradual loss of genetic variation during westward colonization of the Alps

Chromosome number and secondary chromosomal associations in wild populations of Geranium pratense L. from the cold deserts of Lahaul-Spiti (India)

In this work we studied the meiotic chromosome number and details of secondary chromosomal associations recorded for the first time in Geranium pratense L. from the alpine environments in the cold deserts of Lahaul­Spiti (India). All the presently studied individuals of the species existed at 4x level (x = 14). The present chromosome count of n = 28 in the species adds a new cytotype to the already existing diploid chromosome count of 2n = 28 from the Eastern Himalayas and outside of India. Out of the six accessions scored presently four showed normal meiotic course. However, two accessions investigated from Mud, 3800 m and Koksar, 3140 m depicted abnormal meiotic course due to the presence of multivalents and univalents, and secondary associations of bivalents/chromosomes. The secondary chromosomal associations in the species existed among bivalents/chromosomes were noticed in the PMCs at prophase­I (diakinesis) and persisted till the separation of sister chromatids at M­II. The variation in the number of bivalents/chromosomes involved in the secondary associations at M­I (2–8) and A­I/M­II (2–12) has also been recorded. The occurrence of such secondary associations of bivalents/ chromosomes in G. pratense which existed at 4x level indicated the secondary polyploid nature of the species.Впервые изучены мейотическое число хромосом и особенности вторичных хромосомных ассоциаций растений Geranium pratense L., растущих на альпийских высотах холодных пустынь Lahaul-Spiti (Индия). Все изученные особи были 4х (x = 14). Хромосомный набор n = 28 у этого вида добавляет новый цитотип к уже существующему диплоидному хромосомному набору 2n = 28 из Восточных Гималаев и за пределами Индии. Из шести изученных образцов у четырех установлен нормальный ход мейоза. В то же время два образца из районов Mud, 3800 м, и Koksar, 3140 м демонстрировали аномальное течение мейоза из-за наличия мульти- и унивалентов и вторичные ассоциации биваленты/хромосомы. Вторичные хромосомные ассоциации были замечены в профазе І (диакинез) и существовали до разделения сестринских хроматид на стадии M-II. Отмечена также изменчивость числа бивалентов/хромосом, вовлеченных во вторичные ассоциации на стадиях M­I (2–8) и A­I/M­II (2–12). Наличие у G. pratense таких вторичных ассоциаций, которые существуют на уровне 4x, указывает на вторичную полиплоидную природу этого вида.Вперше вивчено мейотичне число хромосом і особливості вторинних хромосомних асоціацій рослин Geranium pratense L., що ростуть на аль-пійських висотах холодних пустель Lahaul-Spiti (Індія. Всі досліджені особини були 4х (х = 14). Хромосомний набірn = 28 у цього виду додає новий цитотип до вже існуючого диплоїдного хромосомного набору n = 28 зі Східних Гімалаїв та за межами Індії. З шести вивчених зразків у чотирьох був нормальний хід мейозу. У той же час два зразки з районів Mud, 3800 м, і Koksar, 3140 м демонстрували аномальний перебіг мейозу через наявність мульти- та унівалентів і вторинні асоціації біваленти/хромосоми. Вторинні хромосомні асоціації спостерігались в профазі І (діакінез) і існували до поділу сестринських хроматид на стадії M-II. Зазначено також мінливість числа бівалентів/хромосом, залучених у вторинні асоціації на стадіях M­I (2–8) і A­I/M­II (2–12). Наявність у G. pratense таких вторинних асоціацій, які існують на рівні 4x, вказує на вторинну поліплоїдну природу цього виду