Phenotyping in field experiments is challenging due to interactions between plants and effects from biotic and abiotic factors which increase complexity in plant development. In such environments, ...visual or destructive measurements are considered the limiting factor and novel approaches are necessary. Remote multispectral imaging is a powerful method that has shown significant potential to estimate crop physiology. However, precise measurements of phenotypic differences between crop varieties in field experiments require exclusion of the disturbances caused by wind and varying sunlight. A mobile and closed multispectral imaging system was developed to study canopies in field experiments. This system shuts out wind and sunlight to ensure the highest possible precision and accuracy. Multispectral images were acquired in an experiment with four different wheat varieties, two different nitrogen levels, replicated on two different soil types at four different dates from 15 May (BBCH 13) to 18 June (BBCH 41 to 57). The images were analyzed and derived vegetation coverage and Normalized Difference Vegetation index (NDVI) were used to assess varietal differences. The results showed potentials for differentiating between the varieties using both vegetation coverage and NDVI, especially at the early growth stages. The perspectives of high-precision and high-throughput imaging for field phenotyping are discussed including the potentials of measuring varietal differences via spectral imaging in comparison to other simpler technologies such as spectral reflectance and RGB imaging.
Leaf senescence is the final stage of leaf development in which the nutrients invested in the leaf are remobilized to other parts of the plant. Whereas senescence is accompanied by a decline in leaf ...cytokinin content, exogenous application of cytokinins or an increase of the endogenous concentration delays senescence and causes nutrient mobilization. The finding that extracellular invertase and hexose transporters, as the functionally linked enzymes of an apolasmic phloem unloading pathway, are coinduced by cytokinins suggested that delay of senescence is mediated via an effect on source-sink relations. This hypothesis was further substantiated in this study by the finding that delay of senescence in transgenic tobacco (Nicotiana tabacum) plants with autoregulated cytokinin production correlates with an elevated extracellular invertase activity. The finding that the expression of an extracellular invertase under control of the senescence-induced SAG12 promoter results in a delay of senescence demonstrates that effect of cytokinins may be substituted by these metabolic enzymes. The observation that an increase in extracellular invertase is sufficient to delay leaf senescence was further verified by a complementing functional approach. Localized induction of an extracellular invertase under control of a chemically inducible promoter resulted in ectopic delay of senescence, resembling the naturally occurring green islands in autumn leaves. To establish a causal relationship between cytokinins and extracellular invertase for the delay of senescence, transgenic plants were generated that allowed inhibition of extracellular invertase in the presence of cytokinins. For this purpose, an invertase inhibitor was expressed under control of a cytokinin-inducible promoter. It has been shown that senescence is not any more delayed by cytokinin when the expression of the invertase inhibitor is elevated. This finding demonstrates that extracellular invertase is required for the delay of senescence by cytokinins and that it is a key element of the underlying molecular mechanism.
is an economically important tree crop widely used for Christmas tree production. After initial growth in nurseries, seedlings are transplanted to the field. Rhizosphere bacterial communities ...generally impact the growth and health of the host plant. However, the dynamics of these communities during
growth in nurseries, and during transplanting, has not previously been addressed. By a 16S rRNA gene amplicon sequencing approach, we characterized the composition and dynamics of bacterial communities in the rhizosphere during early plant growth in field and greenhouse nurseries and for plants transplanted from the greenhouse to the field. Moreover, the N-cycling potential of rhizosphere bacteria across plant age was addressed in both nurseries. Overall, a rhizosphere core microbiome of
, comprising 19.9% of the taxa at genus level, was maintained across plant age, nursery production systems, and even during the transplantation of plants from the greenhouse to the field. The core microbiome included the bacterial genera
,
,
,
,
,
, and
, which harbor several N-fixing and plant growth-promoting taxa. Nevertheless, both plant age and production system caused significant changes in the rhizosphere bacterial communities. Concerning community composition, the relative abundance of Rhizobiales (genera
,
, and
) was higher in the rhizosphere of field-grown
, whereas the relative abundance of Enterobacteriales and Pseudomonadales (genus
) was higher in the greenhouse. Analysis of community dynamics across plant age showed that in the field nursery, the most abundant bacterial orders showed more dynamic changes in their relative abundance in the rhizosphere than in the bulk soil. In the greenhouse, age-dependent dynamics even occurred but affected different taxa than for the field-grown plants. The N-cycling potential of rhizosphere bacterial communities showed an increase of the relative abundance of genes involved in nitrogen fixation and denitrification by plant age. Similarly, the relative abundance of reported nitrogen-fixing or denitrifying bacteria increased by plant age. However, different community structures seemed to lead to an increased potential for nitrogen fixation and denitrification in the field versus greenhouse nurseries.
There is increasing evidence that pathogens do not only elicit direct defense responses, but also cause pronounced changes in primary carbohydrate metabolism. Cell-wall-bound invertases belong to the ...key regulators of carbohydrate partitioning and source-sink relations. Whereas studies have focused so far only on the transcriptional induction of invertase genes in response to pathogen infection, the role of post-translational regulation of invertase activity has been neglected and was the focus of the present study. Expression analyses revealed that the high mRNA level of one out of three proteinaceous invertase inhibitors in source leaves of Arabidopsis thaliana is strongly repressed upon infection by a virulent strain of Pseudomonas syringae pv. tomato DC3000. This repression is paralleled by a decrease in invertase inhibitor activity. The physiological role of this regulatory mechanism is revealed by the finding that in situ invertase activity was detectable only upon infection by P. syringae. In contrast, a high invertase activity could be measured in vitro in crude and cell wall extracts prepared from both infected and non-infected leaves. The discrepancy between the in situ and in vitro invertase activity of control leaves and the high in situ invertase activity in infected leaves can be explained by the pathogen-dependent repression of invertase inhibitor expression and a concomitant reduction in invertase inhibitor activity. The functional importance of the release of invertase from post-translational inhibition for the defense response was substantiated by the application of the competitive chemical invertase inhibitor acarbose. Posttranslational inhibition of extracellular invertase activity by infiltration of acarbose in leaves was shown to increase the susceptibility to P. syringae. The impact of invertase inhibition on spatial and temporal dynamics of the repression of photosynthesis and promotion of bacterial growth during pathogen infection supports a role for extracellular invertase in plant defense. The acarbose-mediated increase in susceptibility was also detectable in sid2 and cpr6 mutants and resulted in slightly elevated levels of salicylic acid, demonstrating that the effect is independent of the salicylic acid-regulated defense pathway. These findings provide an explanation for high extractable invertase activity found in source leaves that is kept inhibited in situ by post-translational interaction between invertase and the invertase inhibitor proteins. Upon pathogen infection, the invertase activity is released by repression of invertase inhibitor expression, thus linking the local induction of sink strength to the plant defense response.
Actinorhizal nodules are structurally different from legume nodules and show a greater similarity to lateral roots. Because of the important role of auxins in lateral root and nodule formation, auxin ...profiles were examined in roots and nodules of the actinorhizal species
Datisca glomerata
and the model legume
Medicago truncatula
. The auxin response in roots and nodules of both species was analyzed in transgenic root systems expressing a beta-glucuronidase gene under control of the synthetic auxin-responsive promoter
DR5
. The effects of two different auxin on root development were compared for both species. The auxin present in nodules at the highest levels was phenylacetic acid (PAA). No differences were found between the concentrations of active auxins of roots
vs.
nodules, while levels of the auxin conjugate indole-3-acetic acid-alanine were increased in nodules compared to roots of both species. Because auxins typically act in concert with cytokinins, cytokinins were also quantified. Concentrations of
cis
-zeatin and some glycosylated cytokinins were dramatically increased in nodules compared to roots of
D. glomerata
, but not of
M. truncatula
. The ratio of active auxins to cytokinins remained similar in nodules compared to roots in both species. The auxin response, as shown by the activation of the
DR5
promoter, seemed significantly reduced in nodules compared to roots of both species, suggesting the accumulation of auxins in cell types that do not express the signal transduction pathway leading to
DR5
activation. Effects on root development were analyzed for the synthetic auxin naphthaleneacetic acid (NAA) and PAA, the dominant auxin in nodules. Both auxins had similar effects, except that the sensitivity of roots to PAA was lower than to NAA. However, while the effects of both auxins on primary root growth were similar for both species, effects on root branching were different: both auxins had the classical positive effect on root branching in
M. truncatula
, but a negative effect in
D. glomerata
. Such a negative effect of exogenous auxin on root branching has previously been found for a cucurbit that forms lateral root primordia in the meristem of the parental root; however, root branching in
D. glomerata
does not follow that pattern.
Tetrapyrrole biosynthesis is controlled by multiple environmental and endogenous cues. Etiolated T-DNA insertion mutants were screened for red fluorescence as result of elevated levels of ...protochlorophyllide and four
red fluorescent in the dark (rfd)
mutants were isolated and identified.
rfd3
and
rfd4
belong to the group of photomorphogenic
cop/det/fus
mutants.
rfd1 and rfd2
had genetic lesions in
RIBA1
and
FLU
encoding the dual-functional protein GTP cyclohydrolase II/3,4-dihydroxy-2-butanone-4-phosphate synthase and a negative regulator of tetrapyrrole biosynthesis, respectively. RIBA1 catalyses the initial reaction of the metabolic pathway of riboflavin biosynthesis and
rfd1
contains reduced contents of riboflavin and the flavo-coenzymes FMN and FAD
.
Transcriptome analysis of
rfd1
revealed up-regulated genes encoding nucleus-localized factors involved in cytokinin signalling and numerous down-regulated
LEA
genes as well as an auxin-inducible
GH3
gene. Alteration of cytokinin metabolism of
rfd1
was confirmed by elevated contents of active forms of cytokinin and stimulated expression of an
ARR6::GUS
reporter construct. An etiolated quadruple
ckx
(cytokinin oxidase) mutant with impaired cytokinin degradation as well as different knockout mutants for the negative AUX/IAA regulators
shy2
-
101 (iaa3)
,
axr2
-
1 (iaa7) and slr
-
1 (iaa14)
showed also excessive protochlorophyllide accumulation. The transcript levels of
CHLH
and
HEMA1
encoding Mg chelatase and glutamyl-tRNA reductase were increased in
rfd1
and the
AUX/IAA
loss-of-function mutants. It is proposed that reduced riboflavin synthesis impairs the activity of the flavin-containing cytokinin oxidase, increases cytokinin contents and de-represses synthesis of 5-aminolevulinic acid of tetrapyrrole metabolism in darkness. As result of the mutant analyses, the antagonistic cytokinin and auxin signalling is required for a balanced tetrapyrrole biosynthesis in the dark.
Physiological studies in plants often require enzyme extraction from tissues containing high concentrations of phenols and polyphenols. Unless removed or neutralized, such compounds may hinder ...extraction, inactivate enzymes, and interfere with enzyme detection. The following protocol for activity assays for enzymes of primary carbohydrate metabolism, while based on our recently published one for quantitative measurement of activities using coupled spectrophotometric assays in a 96-well format, is tailored to the complexities of phenolic- and anthocyanin-rich extracts from grapevine leaf. As a case study we applied the protocol to grapevine leaf samples infected with plant pathogenic bacteria 'Candidatus Phytoplasma solani', known to alter carbohydrate metabolism in grapevine. The described adaptations may be useful for determination of metabolic fingerprints for physiological phenotyping of other plant species with inherently high levels of phenolic compounds.
Summary
Lipid peroxidation may be initiated either by lipoxygenases or by reactive oxygen species (ROS). Enzymatic oxidation of α‐linolenate can result in the biosynthesis of cyclic oxylipins of the ...jasmonate type while free‐radical‐catalyzed oxidation of α‐linolenate may yield several classes of cyclic oxylipins termed phytoprostanes in vivo. Previously, we have shown that one of these classes, the E1‐phytoprostanes (PPE1), occurs ubiquitously in plants. In this work, it is shown that PPE1 are converted to novel cyclopentenone A1‐ and B1‐phytoprostanes (PPA1 and PPB1) in planta. Enhanced formation of PPE1, PPA1, and PPB1 is observed after peroxide stress in tobacco cell cultures as well as after infection of tomato plants with a necrotrophic fungus, Botrytis cinerea. PPA1 and PPB1 display powerful biologic activities including activation of mitogen‐activated protein kinase (MAPK) and induction of glutathione‐S‐transferase (GST), defense genes, and phytoalexins. Data collected so far infer that enhanced phytoprostane formation is a general consequence of oxidative stress in plants. We propose that phytoprostanes are components of an oxidant‐injury‐sensing, archaic signaling system that serves to induce several plant defense mechanisms.
Fructans are polymers of fructose and one of the main constituents of water-soluble carbohydrates in forage grasses and cereal crops of temperate climates. Fructans are involved in cold and drought ...resistance, regrowth following defoliation and early spring growth, seed filling, have beneficial effects on human health and are used for industrial processes. Perennial ryegrass (Lolium perenne L.) serves as model species to study fructan metabolism. Fructan metabolism is under the control of both synthesis by fructosyltransferases (FTs) and breakdown through fructan exohydrolases (FEHs). The accumulation of fructans can be triggered by high sucrose levels and abiotic stress conditions such as drought and cold stress. However, detailed studies on the mechanisms involved in the regulation of fructan metabolism are scarce. Since different phytohormones, especially abscisic acid (ABA), are known to play an important role in abiotic stress responses, the possible short term regulation of the enzymes involved in fructan metabolism by the five classical phytohormones was investigated. Therefore, the activities of enzymes involved in fructan synthesis and breakdown, the expression levels for the corresponding genes and levels for water-soluble carbohydrates were determined following pulse treatments with ABA, auxin (AUX), ethylene (ET), gibberellic acid (GA), or kinetin (KIN). The most pronounced fast effects were a transient increase of FT activities by AUX, KIN, ABA, and ET, while minor effects were evident for 1-FEH activity with an increased activity in response to KIN and a decrease by GA. Fructan and sucrose levels were not affected. This observed discrepancy demonstrates the importance of determining enzyme activities to obtain insight into the physiological traits and ultimately the plant phenotype. The comparative analyses of activities for seven key enzymes of primary carbohydrate metabolism revealed no co-regulation between enzymes of the fructan and sucrose pool.
Extracellular invertase mediates phloem unloading via an apoplastic pathway. The gene encoding isoenzyme Nin88 from tobacco was cloned and shown to be characterized by a specific spatial and temporal ...expression pattern. Tissue-specific antisense repression of Nin88 under control of the corresponding promoter in tobacco results in a block during early stages of pollen development, thus, causing male sterility. This result demonstrates a critical role of extracellular invertase in pollen development and strongly supports the essential function of extracellular sucrose cleavage for supplying carbohydrates to sink tissues via the apoplast. The specific interference with phloem unloading, the sugar status, and metabolic signaling during pollen formation will be a potentially valuable approach to induce male sterility in various crop species for hybrid seed production.
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