Andreas M. Fischer

Senescence, nutrient remobilization, and yield in wheat and barley.

Cereals including wheat and barley are of primary importance to ensure food security for the 21st century. A combination of lab- and field-based approaches has led to a considerably improved understanding of the importance of organ and particularly of whole-plant (monocarpic) senescence for wheat and barley yield and quality. A delicate balance between senescence timing, grain nutrient content, nutrient-use efficiency, and yield needs to be considered to (further) improve cereal varieties for a given environment and end use. The recent characterization of the Gpc-1 (NAM-1) genes in wheat and barley demonstrates the interdependence of these traits. Lines or varieties with functional Gpc-1 genes demonstrate earlier senescence and enhanced grain protein and micronutrient content but, depending on the environment, somewhat reduced yields. A major effort is needed to dissect regulatory networks centred on additional wheat and barley transcription factors and signalling pathways influencing the senescence process. Similarly, while important molecular details of nutrient (particularly nitrogen) remobilization from senescing organs to developing grains have been identified, important knowledge gaps remain. The genes coding for the major proteases involved in senescence-associated plastidial protein degradation are largely unknown. Membrane transport proteins involved in the different transport steps occurring between senescing organ (such as leaf mesophyll) cells and protein bodies in the endosperm of developing grains remain to be identified or further characterized. Existing data suggest that an improved understanding of all these steps will reveal additional, important targets for continued cereal improvement.

Sugars, senescence, and ageing in plants and heterotrophic organisms

Although an involvement of metabolic signals in the regulation of plant senescence has been demonstrated in a range of studies, the exact signalling pathways remain largely unresolved. For leaves, evidence supports a role of sugar accumulation in the initiation and/or acceleration of senescence. However, regulation of senescence or ageing may respond to different metabolic signals in heterotrophic plant organs and heterotrophic organisms. In animals and yeast, dietary restriction results in increased lifespan. In this article, the metabolic regulation of leaf senescence is compared with the effects of dietary restriction. Similarities and differences in the signalling pathways are discussed, including the role of autophagy, TOR (target of rapamycin), Sir2 (silent information regulator-2), and SnRK1 (sucrose non-fermenting-1-related protein kinase-1).

Senescence is accelerated, and several proteases are induced by carbon "feast" conditions in barley (Hordeum vulgare L.) leaves

Leaf senescence is characterized by nitrogen remobilization to developing seeds of annual plants, or surviving organs of perennial species. It has been demonstrated that high carbohydrate levels (carbon “feast”) are associated with the onset of the senescence process. Therefore, the development of model systems allowing the manipulation of leaf carbohydrates constitutes a logical first step in the investigation of processes important during early phases of senescence, such as plastidial protein degradation. In this study, sugar accumulation was induced either by the incubation of excised, mature barley (Hordeum vulgare L.) leaves under relatively strong light, or by the interruption of sieve tubes at the base of the leaf lamina by “steam-girdling”. Accelerated chlorophyll degradation and net proteolysis confirmed successful senescence induction in both model systems, but suggested that girdled leaves are more useful than excised leaves to study proteolysis. Activities or transcript levels of several proteolytic enzymes, including plastidial (aminopeptidases, Clp protease), cytosolic (proteasome) and vacuolar (thiol proteases, an aspartic protease and a serine carboxypeptidase) proteases were clearly induced under these conditions; some of these genes also reacted to other stimuli such as leaf excision. The most interesting finding was the specific induction of a carboxypeptidase gene (cp-mIII) in girdled leaves accumulating high carbohydrate levels. As a previous study from our laboratory, using a genetic approach, has indicated that one or several carboxypeptidases are involved in leaf N remobilization, the detailed characterization of cp-mIII (and, possibly, closely related genes) may considerably improve our understanding of whole-plant N recycling.

The Complex Regulation of Senescence

Everybody is familiar with the visual aspects of plant senescence. Color changes occurring in senescing leaves are, however, only one (albeit striking) symptom of the senescence process. As this process usually leads to cell, tissue, organ or even organism death, its timing is critical for plant fitness and, in an agricultural setting, for crop performance. It is therefore necessary to understand the mechanisms regulating senescence onset and rate, especially at the organ and (for annual species) organism level. Considering the importance of senescence for plant fitness, it is unsurprising that it is influenced by many environmental and genetic factors, rendering its detailed analysis challenging. Numerous molecular and transcriptomic studies have provided us with extensive lists of “senescence-associated genes” or “SAGs,” many of which may have functions in either the regulation or execution of senescence. Functional characterization has been initiated for a subset of SAGs, especially for transcription factors (mainly in the NAC/WRKY families) and protein kinases. A major challenge for senescence research is the efficient integration of available information (consisting of large –omic data sets and detailed single-gene/protein studies) into a more coherent picture, leading to new hypotheses and allowing us to address the most important open questions.

Analysis of barley (Hordeum vulgare) leaf senescence and protease gene expression: a family C1A cysteine protease is specifically induced under conditions characterized by high carbohydrate, but low to moderate nitrogen levels.

SUMMARY Senescence is the highly regulated last developmental phase of plant organs and tissues, and is optimized to allow nutrient remobilization to surviving plant parts, such as seeds of annual crops. High leaf carbohydrate to nitrogen (C : N) ratios have been implicated in the induction or acceleration of the senescence process. *A combination of phloem interruption in mature leaves (by steam-girdling, leading to carbohydrate accumulation from photosynthesis) and varied nitrate supply was used to analyse correlations between metabolite levels, leaf senescence parameters and induction of protease genes and proteolytic activities. *Its strong induction under conditions characterized by high C : N ratios, negative correlation of its transcript levels with chlorophylls and nitrates, its strong induction during developmental leaf senescence and its predicted localization to a lytic vacuolar compartment indicate that, among the genes tested, a family C1A cysteine protease is most likely to participate in bulk protein degradation during barley leaf senescence. *While all the genes analysed were selected based on upregulation during leaf senescence in a previous transcriptomic study, a considerably more detailed picture of protease gene regulation emerged from the data presented here, underlining the usefulness of this experimental approach for further (functional) protease characterization.

Evaluation of Wheat Polyphenol Oxidase Genes

ABSTRACT Polyphenol oxidases (PPOs) present in mature wheat (Triticum aestivum L.) kernels have been implicated in the undesirable darkening of cereal products such as Asian noodles. To accelerate the functional characterization of wheat PPOs and allow the identification of those PPO genes that are primarily involved in food biochemistry, several basic local alignment search tool (BLAST) searches of expressed sequence tag (EST) databases were performed using a known wheat PPO sequence as a search argument; identified ESTs were resequenced and aligned. Results from this study suggest the presence of at least six PPO genes in hexaploid wheat, falling into two clusters with three similar sequences each. Based on the tissues used for cDNA library preparation, three genes (all members of one cluster) are expressed during kernel development and may therefore influence cereal product quality; the remaining three genes (belonging to the second cluster) were isolated from nonkernel cDNA libraries and may not be ex...

A high-grain protein content locus on barley (Hordeum vulgare) chromosome 6 is associated with increased flag leaf proteolysis and nitrogen remobilization

Leaf senescence and nitrogen remobilization from senescing tissues are two important factors determining grain protein content (GPC) in cereals. We compared near-isogenic barley (Hordeum vulgare L.) germplasm varying in the allelic state of a major GPC quantitative trait locus on chromosome 6, delineated by molecular markers HVM74 and ABG458 and explaining approximately 46% of the variability in this trait. High GPC was consistently associated with earlier whole-plant senescence. SDS-PAGE and immunoblot analysis of flag leaf proteins indicated earlier leaf protein [including ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco)] degradation in high-GPC germplasm. This was accompanied by enhanced availability of ammonium and glutamine in developing kernels, suggesting increased phloem retranslocation of nitrogen. Based on previous microarray analysis, we performed a detailed expression study of six leaf genes, tentatively involved in plastidial proteolysis, vacuolar proteolysis, intermediary N metabolism and N transport. All of these were upregulated in high-GPC barley, mostly around 21 to 28 days past anthesis, prior to or around the time demonstrating maximal differences in leaf protein (including Rubisco) levels. Therefore, these genes represent potential targets to manipulate grain protein accumulation. It appears likely that their functional analysis will enhance our understanding of whole-plant N recycling. Additionally, earlier leaf (photosynthetic) protein degradation may lead to reduced N carbon assimilation in high-GPC germplasm, explaining past studies demonstrating a negative correlation between GPC and yield.

Molecular and biochemical characterisation of polyphenol oxidases in developing kernels and senescing leaves of wheat (Triticum aestivum)

Polyphenol oxidases (PPOs) have been implicated in plant defence reactions. From an applied point of view, high PPO activity is associated with browning / darkening of fresh and processed food. Owing to its complex genome and economic importance, wheat (Triticum aestivum L.) represents an interesting system to advance our understanding of plant PPO function. We have previously shown that wheat PPOs are organised in a multigene family, consisting of two distinct phylogenetic clusters with three members each. In this study, we demonstrate that members of one cluster are not expressed in developing kernels or senescing flag leaves. Transcriptional regulation of one major gene in the other cluster largely controls PPO levels in these tissues, at least in the wheat varieties used for this study. Our data further indicate that the product of this gene is present as a latent enzyme during early kernel development, and that the latent enzyme is activated during later developmental phases. Enzyme activation can be achieved in vitro by limited tryptic digestion, but our data do not indicate activation by a proteolytic mechanism in vivo. Together, results presented in this study provide important insights into the regulation of wheat PPO function.

A major grain protein content locus on barley (Hordeum vulgare L.) chromosome 6 influences flowering time and sequential leaf senescence

Timing of various developmental stages including anthesis and whole-plant (‘monocarpic’) senescence influences yield and quality of annual crops. While a correlation between flowering/seed filling and whole-plant senescence has been observed in many annuals, it is unclear how the gene networks controlling these processes interact. Using near-isogenic germplasm, it has previously been demonstrated that a grain protein content (GPC) locus on barley chromosome 6 strongly influences the timing of post-anthesis flag leaf senescence, with high-GPC germplasm senescing early. Here, it is shown that the presence of high-GPC allele(s) at this locus also accelerates pre-anthesis plant development. While floral transition at the shoot apical meristem (SAM; determined by the presence of double ridges) occurred simultaneously, subsequent development was faster in the high- than in the low-GPC line, and anthesis occurred on average 5 d earlier. Similarly, sequential (pre-anthesis) leaf senescence was slightly accelerated, but only after differences in SAM development became visible. Leaf expression levels of four candidate genes (from a list of genes differentially regulated in post-anthesis flag leaves) were much higher in the high-GPC line even before faster development of the SAM became visible. One of these genes may be a functional homologue of Arabidopsis glycine-rich RNA-binding protein 7, which has previously been implicated in the promotion of flowering. Together, the data establish that the GPC locus influences pre- and post-anthesis barley development and senescence, and set the stage for a more detailed analysis of the interactions between the molecular networks controlling these important life history traits.

Effects of a barley (Hordeum vulgare) chromosome 6 grain protein content locus on whole-plant nitrogen reallocation under two different fertilisation regimes

A large fraction of protein N harvested with crop seeds is derived from N remobilisation from senescing vegetative plant parts, while a smaller fraction stems from de novo N assimilation occurring after anthesis. This study contrasts near-isogenic barley (Hordeum vulgare L.) germplasm, varying in the allelic state of a major grain protein content (GPC) locus on chromosome 6. Plant material was grown under both low- and high-N fertilisation levels. The analyses indicated that leaf N remobilisation occurred earlier in high-GPC germplasm under both fertilisation regimes, as indicated by an earlier decrease of total leaf N, chlorophylls, soluble- and membrane-proteins. At the same time, kernel free amino acid levels were enhanced, while leaf free amino acid levels were lower in high-GPC barleys, suggesting enhanced retranslocation of organic N to the developing sinks. Enhanced or longer availability of leaf nitrates was detected in high-GPC varieties and lines, at least under high N fertilisation, indicating that the GPC locus profoundly influences whole-plant N allocation and management. Results presented here, together with data from a recent transcriptomic analysis, make a substantial contribution to our understanding of whole-plant N storage, remobilisation and retranslocation to developing sinks.