SummaryGlucosinolates are a class of plant secondary metabolites that serve as antiherbivore compounds in plant defence. A previously identified Arabidopsis thaliana activation-tagged line, displaying altered levels of secondary metabolites, was shown here to be affected in the content of indolic and aliphatic glucosinolates. The observed chemotype was caused by activation of the R2R3-MYB transcription factor gene HIG1 (HIGH INDOLIC GLUCOSINOLATE 1, also referred to as MYB51). HIG1/MYB51 was shown to activate promoters of indolic glucosinolate biosynthetic genes leading to increased accumulation of indolic glucosinolates. The corresponding loss-of-function mutant hig1-1 contained low levels of glucosinolates. Overexpression of the related transcription factor ATR1/MYB34, which had previously been described as a regulator of indolic glucosinolate and indole-3-acetic acid homeostasis, in the hig1-1 mutant background led to a partial rescue of the mutant chemotype along with a severe high-auxin growth phenotype. Overexpression of MYB122, another close homologue of HIG1/MYB51, did not rescue the hig1-1 chemotype, but caused a high-auxin phenotype and increased levels of indolic glucosinolates in the wild-type. By contrast, overexpression of HIG1/MYB51 resulted in the specific accumulation of indolic glucosinolates without affecting auxin metabolism and plant morphology. Mechanical stimuli such as touch or wounding transiently induced the expression of HIG1/ MYB51 but not of ATR1/MYB34, and HIG1/MYB51 overexpression reduced insect herbivory as revealed by dual-choice assays with the generalist lepidopteran herbivore, Spodoptera exigua. We hypothesize that HIG1/ MYB51 is a regulator of indolic glucosinolate biosynthesis that also controls responses to biotic challenges.

Germin-like proteins (GLPs) have been shown to be encoded by multigene families in several plant species and a role of some subfamily members in defense against pathogen attack has been proposed based on gene regulation studies and transgenic approaches. We studied the function of six GLP subfamilies of barley (Hordeum vulgare) by selecting single mRNAs for gene expression studies as well as overexpression and gene-silencing experiments in barley and Arabidopsis (Arabidopsis thaliana). Expression of all six subfamilies was high in very young seedlings, including roots. The expression pattern gradually changed from developmental to conditional with increasing plant age, whereby pathogen attack and exogenous hydrogen peroxide application were found to be the strongest signals for induction of several GLP subfamilies. Transcripts of four of five GLP subfamilies that are expressed in shoots were predominantly accumulating in the leaf epidermis. Transient overexpression of HvGER4 or HvGER5 as well as transient silencing by RNA interference of HvGER3 or HvGER5 protected barley epidermal cells from attack by the appropriate powdery mildew fungus Blumeria graminis f. sp. hordei. Silencing of HvGER4 induced hypersusceptibility. Transient and stable expression of subfamily members revealed HvGER5 as a new extracellular superoxide dismutase, and protection by overexpression could be demonstrated to be dependent on superoxide dismutase activity of the encoded protein. Data suggest a complex interplay of HvGER proteins in fine regulation of basal resistance against B. graminis.

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Hybrid breeding promises to boost yield and stability. The single most important element in implementing hybrid breeding is the recognition of a high-yielding heterotic pattern. We have developed a three-step strategy for identifying heterotic patterns for hybrid breeding comprising the following elements. First, the full hybrid performance matrix is compiled using genomic prediction. Second, a high-yielding heterotic pattern is searched based on a developed simulated annealing algorithm. Third, the long-term success of the identified heterotic pattern is assessed by estimating the usefulness, selection limit, and representativeness of the heterotic pattern with respect to a defined base population. This three-step approach was successfully implemented and evaluated using a phenotypic and genomic wheat dataset comprising 1,604 hybrids and their 135 parents. Integration of metabolomic-based prediction was not as powerful as genomic prediction. We show that hybrid wheat breeding based on the identified heterotic pattern can boost grain yield through the exploitation of heterosis and enhance recurrent selection gain. Our strategy represents a key step forward in hybrid breeding and is relevant for self-pollinating crops, which are currently shifting from pure-line to high-yielding and resilient hybrid varieties.hybrid breeding | genomic prediction | heterotic pattern

Barley (Hordeum vulgare) grains synthesize starch as the main storage compound. However, some starch is degraded already during caryopsis development. We studied temporal and spatial expression patterns of genes coding for enzymes of starch synthesis and degradation. These profiles coupled with measurements of selected enzyme activities and metabolites have allowed us to propose a role for starch degradation in maternal and filial tissues of developing grains. Early maternal pericarp functions as a major short-term starch storage tissue, possibly ensuring sink strength of the young caryopsis. Gene expression patterns and enzyme activities suggest two different pathways for starch degradation in maternal tissues. One pathway possibly occurs via a-amylases 1 and 4 and b-amylase 1 in pericarp, nucellus, and nucellar projection, tissues that undergo programmed cell death. Another pathway is deducted for living pericarp and chlorenchyma cells, where transient starch breakdown correlates with expression of chloroplast-localized b-amylases 5, 6, and 7, glucan, water dikinase 1, phosphoglucan, water dikinase, isoamylase 3, and disproportionating enzyme. The suite of genes involved in starch synthesis in filial starchy endosperm is much more complex than in pericarp and involves several endosperm-specific genes. Transient starch turnover occurs in transfer cells, ensuring the maintenance of sink strength in filial tissues and the reallocation of sugars into more proximal regions of the starchy endosperm. Starch is temporally accumulated also in aleurone cells, where it is degraded during the seed filling period, to be replaced by storage proteins and lipids.

Recent advances in instrumentation and sample preparation have facilitated the mass spectrometric (MS) imaging of a large variety of biological molecules from small metabolites to large proteins. The technique can be applied at both the tissue and the single-cell level, and provides information regarding the spatial distribution of specific molecules. Nevertheless, the use of MS imaging in plant science remains far from routine, and there is still a need to adapt protocols to suit specific tissues. We present an overview of MALDI-imaging MS (MSI) technology and its use for the analysis of plant tissue. Recent methodological developments have been summarized, and the major challenges involved in using MALDI-MSI, including sample preparation, the analysis of metabolites and peptides, and strategies for data evaluation are all discussed. Some attention is given to the identification of differentially distributed compounds. To date, the use of MALDI-MSI in plant research has been limited. Examples include leaf surface metabolite maps, the characterization of soluble metabolite translocation in planta, and the profiling of protein/metabolite patterns in cereal grain cross-sections. Improvements to both sample preparation strategies and analytical platforms (aimed at both spectrum acquisition and post-acquisition analysis) will enhance the relevance of MALDI-MSI technology in plant research.

Protoporphyrinogen IX oxidase is the last enzyme in the common pathway of heme and chlorophyll synthesis and provides precursor for the mitochondrial and plastidic heme synthesis and the predominant chlorophyll synthesis in plastids. We cloned two different, full-length tobacco cDNA sequences by complementation of the protoporphyrin-IX-accumulating Escherichia coli hemG mutant from heme auxotrophy. The two sequences show similarity to the recently published Arabidopsis PPOX, Bacillus subtilis hemY, and to mammalian sequences encoding protoporphyrinogen IX oxidase. One cDNA sequence encodes a 548-amino acid residues protein with a putative transit sequence of 50 amino acid residues, and the second cDNA encodes a protein of 504 amino acid residues. Both deduced protein sequences share 27.2% identical amino acid residues. The first in vitro translated protoporphyrinogen IX oxidase could be translocated to plastids, and the approximately 53-kDa mature protein was detected in stroma and membrane fraction. The second enzyme was targeted to mitochondria without any detectable reduction in size. Localization of both enzymes in subcellular fractions was immunologically confirmed. Steadystate RNA analysis indicates an almost synchronous expression of both genes during tobacco plant development, greening of young seedlings, and diurnal and circadian growth. The mature plastidal and the mitochondrial isoenzyme were overexpressed in E. coli. Bacterial extracts containing the recombinant mitochondrial enzyme exhibit high protoporphyrinogen IX oxidase activity relative to control strains, whereas the plastidal enzyme could only be expressed as an inactive peptide. The data presented confirm a compartmentalized pathway of tetrapyrrole synthesis with protoporphyrinogen IX oxidase in plastids and mitochondria.

Heterosis is defined as the increased vigour of hybrids in comparison to their parents. We investigated 24 F1 hybrid lines of Arabidopsis thaliana generated by reciprocally crossing either C24 or Col with six other parental accessions (Can, Co, Cvi, Ler, Rsch, Te) that differ widely in their freezing tolerance. The crosses differed in the degree of heterosis for freezing tolerance, both in the non-acclimated state and after a 14 d cold acclimation period. Crosses with C24 showed more heterosis than crosses with Col, and heterosis was stronger in acclimated than in non-acclimated plants. Leaf content of soluble sugars and proline showed more deviation from mid-parent values in crosses involving C24 than in those involving Col, and deviations were larger in acclimated than in non-acclimated plants. There were significant correlations between the content of different sugars and leaf freezing tolerance, as well as between heterosis effects in freezing tolerance and sugar content. Flavonoid content and composition varied between accessions, and between non-acclimated and acclimated plants. In the crosses, large deviations from the mid-parent values in the contents of different flavonols occurred, and there were strikingly strong correlations between both flavonol content and freezing tolerance, and between heterosis effects in freezing tolerance and flavonol content.