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Chloroplasts need to import preproteins and amino acids from the cytosol during their light-induced differentiation. Similarly, chloroplasts have to export organic matter including proteins and amino acids during leaf senescence. Members of the PRAT (preprotein and amino acid transporter) family are candidate transporters for both processes. Here, we defined the role of two small PRAT gene families, At4g26670 and At5g55510 (HP20 subfamily) versus At3g49560 and At5g24650 (HP30 subfamily) during greening of etiolated plants and during leaf senescence. Using a combination of reverse genetics, protein biochemistry and physiological tools, evidence was obtained for a role of chloroplast HP20, HP30 and HP30-2 in protein, but not amino acid, import into chloroplasts. HP20, HP30 and HP30-2 form larger complexes involved in the uptake of transit sequence-less cytosolic precursors. In addition, we identified a fraction of HP30-2 in mitochondria where it served a similar function as found for chloroplasts and operated in the uptake of transit sequence-less cytosolic precursor proteins. By contrast, HP22 was found to act in the export of proteins from chloroplasts during leaf senescence, and thus its role is entirely different from that of its orthologue, HP20. HP22 is part of a unique protein complex in the envelope of senescing chloroplasts that comprises at least 11 proteins and contains with HP65b (At5g55220) a protein that is related to the bacterial trigger factor chaperone. An ortholog of HP65b exists in the cyanobacterium Synechocystis and has previously been implicated in protein secretion. Whereas plants depleted of either HP22 or HP65b or even both were increasingly delayed in leaf senescence and retained much longer stromal chloroplast constituents than wild-type plants, HP22 overexpressors showed premature leaf senescence that was associated with accelerated losses of stromal chloroplast proteins. Together, our results identify the PRAT protein family as a unique system for importing and exporting proteins from chloroplasts.

Aminoacyl-tRNA synthetases (AaRS) charge tRNAs with amino acids for protein translation. In plants, cytoplasmic, mitochondrial, and chloroplast AaRS exist that are all coded for by nuclear genes and must be imported from the cytosol. In addition, only a few of the mitochondrial tRNAs needed for translation are encoded in mitochondrial DNA. Despite considerable progress made over the last few years, still little is known how the bulk of cytosolic AaRS and respective tRNAs are transported into mitochondria. Here, we report the identification of a protein complex that ties AaRS and tRNA import into the mitochondria of Arabidopsis thaliana. Using leucyl-tRNA synthetase 2 (LeuRS2) as a model for a mitochondrial signal peptide (MSP)-less precursor, a ≈30 kDa protein was identified that interacts with LeuRS2 during import. The protein identified is identical with a previously characterized mitochondrial protein designated HP30-2 (encoded by At3g49560) that contains a sterile alpha motif (SAM) similar to that found in RNA binding proteins. HP30-2 is part of a larger protein complex that contains with TIM22, TIM8, TIM9 and TIM10 four previously identified components of the translocase for MSP-less precursors. Lack of HP30-2 perturbed mitochondrial biogenesis and function and caused seedling lethality during greening, suggesting an essential role of HP30-2 in planta.

There are specific advantages of using microspores as explants: (1) A small number of explant donors are required to obtain the desired number of pollen embryoids for genetic transformation and (2) microspores constitute a synchronous mass of haploid cells, which are transformable by various means and convertible to doubled haploids therefore allow production of homozygous genotypes in a single generation. Additionally, it has been demonstrated in wheat and other crops that microspores can be easily induced to produce embryoids and biolistic approach to produce a large number of transformants. In view of these listed advantages, we optimized the use of microspore-derived calli for biolistic transformation of wheat. The procedure takes about 6 months to obtain the viable transformants in the spring wheat background. In the present communication, we demonstrated the use of this method to produce the reduced immunogenicity wheat genotypes.

The present and subsequent chapters in this volume are dedicated to the life and research of Professor Diter von Wettstein who contributed immensely to the development of science and education. His contributions spanned various fields of science such as genetics, physiology, ultrastructural analysis, molecular biology, genomics, and biotechnology including genome editing. He performed and promoted pioneering research in the fields of epigenetics, directed evolution of enzymes, synthetic biology (promoter and gene optimizations), and genomics (genome sequencing of baker's yeast). Glimpses of his time at the Carlsberg Laboratory and Washington State University, with examples from the research performed at these institutions, are included in this chapter. His life is an inspiration to the next generation of biologists. Despite difficult situations, his persistent efforts and keen desire to learn enabled him to overcome obstacles. He always tried to attain the best, excelling in translating fundamental knowledge into applications.

Ubiquitous nature of prolamin proteins dubbed gluten from wheat and allied cereals imposes a major challenge in the treatment of celiac disease, an autoimmune disorder with no known treatment other than abstinence diet. Administration of hydrolytic glutenases as food supplement is an alternative to deliver the therapeutic agents directly to the small intestine, where sensitization of immune system and downstream reactions takes place. The aim of the present research was to evaluate the capacity of wheat grain to express and store hydrolytic enzymes capable of gluten detoxification. For this purpose, wheat scutellar calli were biolistically transformed to generate plants expressing a combination of glutenase genes for prolamin detoxification. Digestion of prolamins with barley endoprotease B2 (EP-HvB2) combined with Flavobacterium meningosepticum prolyl endopeptidase (PE-FmPep) or Pyrococcus furiosus prolyl endopeptidase (PE-PfuPep), significantly reduced (up to 67%) the amount of the indigestible gluten peptides of all prolamin families tested. Seven of the 168 generated lines showed inheritance of transgene to the T(2) generation. Reversed phase high performance liquid chromatography of gluten extracts under simulated gastrointestinal conditions allowed the identification of five T(2) lines that contained significantly reduced amounts of immunogenic, celiac disease-provoking gliadin peptides. These findings were complemented by the R5 ELISA test results where up to 72% reduction was observed in the content of immunogenic peptides. The developed wheat genotypes open new horizons for treating celiac disease by an intraluminal enzyme therapy without compromising their agronomical performance.

Chloroplasts and mitochondria contain a family of putative preprotein and amino acid transporters designated PRAT. Here, we analyzed the role of two previously characterized PRAT protein family members, encoded by At3g49560 (HP30) and At5g24650 (HP30-2), in planta using a combination of genetic, cell biological and biochemical approaches. Expression studies and green fluorescent protein tagging identified HP30-2 both in chloroplasts and mitochondria, whereas HP30 was located exclusively in chloroplasts. Biochemical evidence was obtained for an association of mitochondrial HP30-2 with two distinct protein complexes, one containing the inner membrane translocase TIM22 and the other containing an alternative NAD(P)H dehydrogenase subunit (NDC1) implicated in a respiratory complex 1-like electron transport chain. Through its association with TIM22, HP30-2 is involved in the uptake of carrier proteins and other, hydrophobic membrane proteins lacking cleavable NH2 -terminal presequences, whereas HP30-2's interaction with NDC1 may permit controlling mitochondrial biogenesis and activity.

There are specific advantages of using microspores as explants: (1) A small number of explant donors are required to obtain the desired number of pollen embryoids for genetic transformation and (2) microspores constitute a synchronous mass of haploid cells, which are transformable by various means and convertible to doubled haploids therefore allow production of homozygous genotypes in a single generation. Additionally, it has been demonstrated in wheat and other crops that microspores can be easily induced to produce embryoids and biolistic approach to produce a large number of transformants. In view of these listed advantages, we optimized the use of microspore-derived calli for biolistic transformation of wheat. The procedure takes about 6 months to obtain the viable transformants in the spring wheat background. In the present communication, we demonstrated the use of this method to produce the reduced immunogenicity wheat genotypes.

Together with the interaction of miR319-regulated TCP transcription factors and the COI1 receptor, the formation of LOX2-AOS-AOC2 complexes controls key steps in jasmonic acid precursor biosynthesis, facilitating fine-tuning of the plant’s defense machinery., Oxygenated membrane fatty acid derivatives termed oxylipins play important roles in plant defense against biotic and abiotic cues. Plants challenged by insect pests, for example, synthesize a blend of different defense compounds that include volatile aldehydes and jasmonic acid (JA), among others. Because all oxylipins are derived from the same pathway, we investigated how their synthesis might be regulated, focusing on two closely related atypical cytochrome P450 enzymes designated CYP74A and CYP74B, respectively, allene oxide synthase (AOS) and hydroperoxide lyase (HPL). These enzymes compete for the same substrate but give rise to different products: the final product of the AOS branch of the oxylipin pathway is JA, while those of the HPL branch comprise volatile aldehydes and alcohols. AOS and HPL are plastid envelope enzymes in Arabidopsis thaliana but accumulate at different locations. Biochemical experiments identified AOS as a constituent of complexes also containing lipoxygenase 2 (LOX2) and allene oxide cyclase (AOC), which catalyze consecutive steps in JA precursor biosynthesis, while excluding the concurrent HPL reaction. Based on published X-ray data, the structure of this complex was modelled and amino acids involved in catalysis and subunit interactions predicted. Genetic studies identified the microRNA 319-regulated clade of TCP (TEOSINTE BRANCHED/CYCLOIDEA/PCF) transcription factor genes and CORONATINE INSENSITIVE 1 (COI1) as controlling JA production through the LOX2-AOS-AOC2 complex. Together, our results define a molecular branch point in oxylipin biosynthesis that allows fine-tuning of the plant’s defense machinery in response to biotic and abiotic stimuli.

Plants have evolved an intricate regulatory network of proteases and corresponding protease inhibitors (PI), which operate in various biological pathways and serve diverse spatiotemporal functions during the sedentary life of a plant. Intricacy of the regulatory network can be anticipated from the observation that, depending on the developmental stage and environmental cue(s), either a single PI or multiple PIs regulate the activity of a given protease. On the other hand, the same PI often interacts with different targets at different places, necessitating another level of fine control to be added in planta. Here, it is reported on how the activity of a papain-like cysteine protease dubbed RD21 (RESPONSIVE TO DESICCATION 21) is differentially regulated by serpin and Kunitz PIs over plant development and how this mechanism contributes to defenses against herbivorous arthropods and microbial pests.

Nitric oxide (NO) is a signaling molecule that participates at physiological concentrations in diverse regulatory pathways. However, when NO concentration reaches levels higher than normal, it is sensed as a stress, which results in epigenetic alterations and chromatin remodeling in plants. Here, we showed that exposure to 0.5 mM of sodium nitroprusside (SNP), a NO donor, induces stress symptoms in rice seedlings that result in complete growth inhibition. Moreover, these stress symptoms in SNP-treated plants were found to be associated with methylation changes in genomic DNA, which predominantly involve hypomethylation at the CHG sites. Concomitant with these changes, transcriptional activation of a number of genes and transposable elements (TEs) was also observed. Further analysis revealed that expression of four genes, Chromomethylase 3 (OsCMT3), deficient in DNA methylation 1 (OsDDM1a), OsDDM1b, and DEMETER (OsDME), involved in chromatin remodeling and DNA methylation homoeostasis have been specifically perturbed.

Plants have evolved an intricate regulatory network of proteases and corresponding protease inhibitors (PI), which operate in various biological pathways and serve diverse spatiotemporal functions during the sedentary life of a plant. Intricacy of the regulatory network can be anticipated from the observation that, depending on the developmental stage and environmental cue(s), either a single PI or multiple PIs regulate the activity of a given protease. On the other hand, the same PI often interacts with different targets at different places, necessitating another level of fine control to be added in planta. Here, it is reported on how the activity of a papain-like cysteine protease dubbed RD21 (RESPONSIVE TO DESICCATION 21) is differentially regulated by serpin and Kunitz PIs over plant development and how this mechanism contributes to defenses against herbivorous arthropods and microbial pests.

Phosphite (Phi) is used commercially to manage diseases mainly caused by oomycetes, primarily due to its low cost compared with other fungicides and its persistent control of oomycetous pathogens. We explored the use of Phi in controlling the fungal pathogens Puccinia emaculata and Phakopsora pachyrhizi, the causal agents of switchgrass rust and Asian soybean rust, respectively. Phi primes host defenses and efficiently inhibits the growth of P. emaculata, P. pachyrhizi and several other fungal pathogens tested. To understand these Phi-mediated effects, a detailed molecular analysis was undertaken in both the host and the pathogen. Transcriptomic studies in switchgrass revealed that Phi activates plant defense signaling as early as 1 h after application by increasing the expression of several cytoplasmic and membrane receptor-like kinases and defense-related genes within 24 h of application. Unlike in oomycetes, RNA sequencing of P. emaculata and P. pachyrhizi did not exhibit Phi-mediated retardation of cell wall biosynthesis. The genes with reduced expression in either or both rust fungi belonged to functional categories such as ribosomal protein, actin, RNA-dependent RNA polymerase, and aldehyde dehydrogenase. A few P. emaculata genes that had reduced expression upon Phi treatment were further characterized. Application of double-stranded RNAs specific to P. emaculata genes encoding glutamate N-acetyltransferase and cystathionine gamma-synthase to switchgrass leaves resulted in reduced disease severity upon P. emaculata inoculation, suggesting their role in pathogen survival and/or pathogenesis.

Chloroplasts and mitochondria contain a family of putative preprotein and amino acid transporters designated PRAT. Here, we analyzed the role of two previously characterized PRAT protein family members, encoded by At3g49560 (HP30) and At5g24650 (HP30-2), in planta using a combination of genetic, cell biological and biochemical approaches. Expression studies and green fluorescent protein tagging identified HP30-2 both in chloroplasts and mitochondria, whereas HP30 was located exclusively in chloroplasts. Biochemical evidence was obtained for an association of mitochondrial HP30-2 with two distinct protein complexes, one containing the inner membrane translocase TIM22 and the other containing an alternative NAD(P)H dehydrogenase subunit (NDC1) implicated in a respiratory complex 1-like electron transport chain. Through its association with TIM22, HP30-2 is involved in the uptake of carrier proteins and other, hydrophobic membrane proteins lacking cleavable NH

Proteolytic enzymes (proteases) participate in a vast range of physiological processes, ranging from nutrient digestion to blood coagulation, thrombosis, and beyond. In plants, proteases are implicated in host recognition and pathogen infection, induced defense (immunity), and the deterrence of insect pests. Because proteases irreversibly cleave peptide bonds of protein substrates, their activity must be tightly controlled in time and space. Here, we report an example of how nature evolved alternative mechanisms to fine-tune the activity of a cysteine protease dubbed RD21 (RESPONSIVE TO DESICCATION-21). One mechanism in the model plant Arabidopsis thaliana studied here comprises irreversible inhibition of RD21’s activity by Serpin1, whereas the other mechanism is a result of the reversible inhibition of RD21 activity by a Kunitz protease inhibitor named water-soluble chlorophyll-binding protein (WSCP). Activity profiling, complex isolation, and homology modeling data revealed unique interactions of RD21 with Serpin1 and WSCP, respectively. Expression studies identified only partial overlaps in Serpin1 and WSCP accumulation that explain how RD21 contributes to the innate immunity of mature plants and arthropod deterrence of seedlings undergoing skotomorphogenesis and greening.

Although studied extensively since 1958, the molecular mode of action of the Pairing homeologous 1 (Ph1) gene is still unknown. In polyploid wheat, the diploid-like chromosome pairing is principally controlled by the Ph1 gene via preventing homeologous chromosome pairing (HECP). Here, we report a candidate Ph1 gene (C-Ph1) present in the Ph1 locus, transient as well as stable silencing of which resulted in a phenotype characteristic of the Ph1 gene mutants, including HECP, multivalent formation, and disrupted chromosome alignment on the metaphase I (MI) plate. Despite a highly conserved DNA sequence, the C-Ph1 gene homeologues showed a dramatically different structure and expression pattern, with only the 5B copy showing MI-specific expression, further supporting our claim for the Ph1 gene. In agreement with the previous reports about the Ph1 gene, the predicted protein of the 5A copy of the C-Ph1 gene is truncated, and thus perhaps less effective. The 5D copy is expressed around the onset of meiosis; thus, it may function during the earlier stages of chromosome pairing. Along with alternate splicing, the predicted protein of the 5B copy is different from the protein of the other two copies because of an insertion. These structural and expression differences among the homeologues concurred with the previous observations about Ph1 gene function. Stable RNAi silencing of the wheat gene in Arabidopsis showed multivalents and centromere clustering during meiosis I.

Two closely related genes encoding the jasmonate-induced protein 60 (JIP60) were identified in the barley genome. The gene on chromosome arm 4HL encodes the previously identified protein encoded by the cDNA X66376.1. This JIP60 protein is characterized here and shown to consist of two domains: an NH2-terminal domain related to ribosome-inactivating proteins and a COOH-terminal domain, which displays similarity to eukaryotic translation initiation factor 4E (eIF4E). JIP60 undergoes processing in vivo, as a result of which JIP60's COOH-terminal eIF4E domain is released and functions in recruiting a subset of cellular messengers for translation. This effect was observed for both MeJA-treated and naturally senescing plants. Because the JIP60 gene is in close proximity to several quantitative trait loci for both biotic and abiotic stress resistance, our results identify a unique target for future breeding programs.

Hexaploid bread wheat (Triticum aestivum L., genome BBAADD) is generally more salt tolerant than its tetraploid wheat progenitor (Triticum turgidum L.). However, little is known about the physiological basis of this trait or about the relative contributions of allohexaploidization and subsequent evolutionary genetic changes on the trait development. Here, we compared the salt tolerance of a synthetic allohexaploid wheat (neo-6x) with its tetraploid (T. turgidum; BBAA) and diploid (Aegilops tauschii; DD) parents, as well as a natural hexaploid bread wheat (nat-6x). We studied 92 morphophysiological traits and analyzed homeologous gene expression of a major salt-tolerance gene High-Affinity K(+) Transporter 1;5 (HKT1;5). We observed that under salt stress, neo-6x exhibited higher fitness than both of its parental genotypes due to inheritance of favorable traits like higher germination rate from the 4x parent and the stronger root Na(+) retention capacity from the 2x parent. Moreover, expression of the D-subgenome HKT1;5 homeolog, which is responsible for Na(+) removal from the xylem vessels, showed an immediate transcriptional reprogramming following allohexaploidization, i.e., from constitutive high basal expression in Ae. tauschii (2x) to salt-induced expression in neo-6x. This phenomenon was also witnessed in the nat-6x. An integrated analysis of 92 traits showed that, under salt-stress conditions, neo-6x resembled more closely the 2x than the 4x parent, suggesting that the salt stress induces enhanced expressivity of the D-subgenome homeologs in the synthetic hexaploid wheat. Collectively, the results suggest that condition-dependent functionalization of the subgenomes might have contributed to the wide-ranging adaptability of natural hexaploid wheat.

Wheat prolamins, commonly known as “gluten”, are a complex mixture of 71–78 proteins, which constitute ~80% of the proteins in the wheat grains and supply 50% of the global dietary protein demand. Prolamins are also responsible for numerous gluten-induced disorders and determine the unique visco-elastic properties of the wheat dough. These properties necessitate the reliable determination of the prolamin composition in wheat grains and their derived products. Therefore, this study examined the impact of HPLC conditions, including column type, column temperature, flow rate, and the gradient of polar and non-polar solvents in the mobile phase, to improve the analytical resolution of prolamins. The following conditions were found optimal for analyses: column temperature 60 °C, flow rate 1.0 mL/min and an elution gradient of 20%–60% of 0.1% trifluoroacetic acid + acetonitrile in 60 min. For further improvement of resolution, gliadin and glutenin extracts were analyzed using MALDI-TOF-MS in combination with HPLC fractionation. Two semi-quantitative methods, densitometry of stained polyacrylamide gels and HPLC, were used to determine relative prolamin quantities and the correspondence between the methods was established. The combinatorial gluten analyses approach developed during the present study was used to analyze prolamin profiles of wheat transformants expressing DEMETER silencing artificial microRNA, and the results are discussed.

The channeling of metabolites is an essential step of metabolic regulation in all living organisms. Multifunctional enzymes with defined domains for metabolite compartmentalization are rare, but in many cases, larger assemblies forming multimeric protein complexes operate in defined metabolic shunts. In Arabidopsis thaliana, a multimeric complex was discovered that contains a 13-lipoxygenase and allene oxide synthase (AOS) as well as allene oxide cyclase. All three plant enzymes are localized in chloroplasts, contributing to the biosynthesis of jasmonic acid (JA). JA and its derivatives act as ubiquitous plant defense regulators in responses to both biotic and abiotic stresses. AOS belongs to the superfamily of cytochrome P450 enzymes and is named CYP74A. Another CYP450 in chloroplasts, hydroperoxide lyase (HPL, CYP74B), competes with AOS for the common substrate. The products of the HPL reaction are green leaf volatiles that are involved in the deterrence of insect pests. Both enzymes represent non-canonical CYP450 family members, as they do not depend on O2 and NADPH-dependent CYP450 reductase activities. AOS and HPL activities are crucial for plants to respond to different biotic foes. In this mini-review, we aim to summarize how plants make use of the LOX2−AOS−AOC2 complex in chloroplasts to boost JA biosynthesis over volatile production and how this situation may change in plant communities during mass ingestion by insect pests.

A family of 17 putative preprotein and amino acid transporters designated PRAT has been identified in Arabidopsis thaliana, comprising PRAT proteins in mitochondria and chloroplasts. Although some PRAT proteins, such as the translocon of the mitochondrial inner membrane (TIM) proteins TIM22 and TIM23, play decisive roles for the translocation and import of mitochondrial inner membrane proteins, little is known about the role of the different PRAT members in chloroplasts. Here we report the identification of three distinct PRAT proteins as part of a unique protein import site. One of the identified PRAT proteins is identical with a previously characterized hypothetical protein (HP) of 20 kDa designated HP20 of the outer plastid envelope membrane. The second PRAT component is represented by HP30, and the third is identical to HP30-2, a close relative of HP30. Both HP30 and HP30-2 are inner plastid envelope membrane proteins of chloroplasts. Using biochemical, cell biological, and genetic approaches we demonstrate that all three PRAT proteins cooperate during import of transit sequence-less proteins, such as the quinone oxidoreductase homolog ceQORH used as model, into the inner chloroplast envelope membrane. Our data are reminiscent of findings reported for the TIM22 translocase, which is involved in the import of carrier proteins and other, hydrophobic membrane proteins lacking cleavable transit sequences into the inner mitochondrial membrane. Together our results establish the PRAT family as a widely used system of protein translocases in different membranes of endosymbiotic origin.

Polyploidy or whole-genome duplication is recurrent in plant evolution, yet only a small fraction of whole-genome duplications has led to successful speciation. A major challenge in the establishment of nascent polyploids is sustained karyotype instability, which compromises fitness. The three putative diploid progenitors of bread wheat, with AA, SS (S ∼ B), and DD genomes occurred sympatrically, and their cross-fertilization in different combinations may have resulted in fertile allotetraploids with various genomic constitutions. However, only SSAA or closely related genome combinations have led to the speciation of tetraploid wheats like Triticum turgidum and Triticum timopheevii. We analyzed early generations of four newly synthesized allotetraploid wheats with genome compositions S(sh)S(sh)A(m)A(m), S(l)S(l)AA, S(b)S(b)DD, and AADD by combined fluorescence and genomic in situ hybridization-based karyotyping. Results of karyotype analyses showed that although S(sh)S(sh)A(m)A(m) and S(l)S(l)AA are characterized by immediate and persistent karyotype stability, massive aneuploidy and extensive chromosome restructuring are associated with S(b)S(b)DD and AADD in which parental subgenomes showed markedly different propensities for chromosome gain/loss and rearrangements. Although compensating aneuploidy and reciprocal translocation between homeologs prevailed, reproductive fitness was substantially compromised due to chromosome instability. Strikingly, localized genomic changes in repetitive DNA and copy-number variations in gene homologs occurred in both chromosome stable lines, S(sh)S(sh)A(m)A(m) and S(l)S(l)AA. Our data demonstrated that immediate and persistent karyotype stability is intrinsic to newly formed allotetraploid wheat with genome combinations analogous to natural tetraploid wheats. This property, coupled with rapid gene copy-number variations, may have laid the foundation of tetraploid wheat establishment.

Microspores are preferred explant choice for genetic transformation, as their use shortens the duration of obtaining homozygous transformants. All established gene-delivery methods of particle bombardment, electroporation, and cocultivation with Agrobacterium tumefaciens were optimized on androgenic microspores or derived tissues. In the biolistic gene delivery method 35-40 days old haploid microspore embryoids were used for genetic transformation, whereas freshly isolated androgenic microspores were used for genetic transformation in the electroporation and Agrobacterium cocultivation-based methods. The genetic transformation methods of biolistic gene-delivery and electroporation gave rise to the chimeric plants, whereas the method involving cocultivation with Agrobacterium yielded homozygous transformants. These methods were tested on a large number of cultivars belonging to different market classes of wheat, and found to be fairly independent of the explant genotype. Other benefits of using microspores or derived tissues for transformation are: (1) a few explant donors are required to obtain desired transformants and (2) the time required for obtaining homozygous transformants is about 8 months in case of spring wheat genotypes and about a year in case of winter wheat genotypes.

NADPH:protochlorophyllide oxidoreductase (POR) is a key enzyme for the light-induced greening of etiolated angiosperm plants. It belongs to the 'RED' family of reductases, epimerases and dehydrogenases. All POR proteins characterized so far contain evolutionarily conserved cysteine residues implicated in protochlorophyllide (Pchlide)-binding and catalysis. cDNAs were constructed by site-directed mutagenesis that encode PORB mutant proteins with defined Cys→Ala exchanges. These cDNAs were expressed in transgenic plants of a PORB-deficient knock-out mutant (porB) of Arabidopsis thaliana. Results show that porB plants expressing PORB mutant proteins with Ala substitutions of Cys276 or Cys303 are hypersensitive to high-light conditions during greening. Hereby, failure to assemble higher molecular weight complexes of PORB with its twin isoenzyme, PORA, as encountered with (Cys303→Ala)-PORB plants, caused more severe effects than replacing Cys276 by an Ala residue in the active site of the enzyme, as encountered in (Cys276→Ala)-PORB plants. Our results are consistent with the presence of two distinct pigment binding sites in PORB, with Cys276 establishing the active site of the enzyme and Cys303 providing a second, low affinity pigment binding site that is essential for the assembly of higher molecular mass light-harvesting PORB::PORA complexes and photoprotection of etiolated seedlings. Failure to assemble such complexes provoked photodynamic damage through the generation of singlet oxygen. Together, our data highlight the importance of PORB for Pchlide homoeostasis and greening in Arabidopsis.

Seed predators can cause mass ingestion of larger seed populations. As well, herbivorous arthropods attempt to attack etiolated seedlings and chose the apical hook for ingestion, aimed at dropping the cotyledons for later consumption. Etiolated seedlings, as we show here, have established an efficient mechanism of protecting their Achilles' heel against these predators, however. Evidence is provided for a role of jasmonic acid (JA) in this largely uncharacterized plant-herbivore interaction during skotomorphogenesis and that this comprises the temporally and spatially tightly controlled synthesis of a cysteine protease inhibitors of the Kunitz family. Interestingly, the same Kunitz protease inhibitor was found to be expressed in flowers of Arabidopsis where endogenous JA levels are high for fertility. Because both the apical hook and inflorescences were preferred isopod targets in JA-deficient plants that could be rescued by exogenously administered JA, our data identify a JA-dependent mechanism of plant arthropod deterrence that is recalled in different organs and at quite different times of plant development.

Leaf senescence is the terminal stage in the development of perennial plants. Massive physiological changes occur that lead to the shut down of photosynthesis and a cessation of growth. Leaf senescence involves the selective destruction of the chloroplast as the site of photosynthesis. Here, we show that 13-lipoxygenase (13-LOX) accomplishes a key role in the destruction of chloroplasts in senescing plants and propose a critical role of its NH2-terminal chloroplast transit peptide. The 13-LOX enzyme identified here accumulated in the plastid envelope and catalyzed the dioxygenation of unsaturated membrane fatty acids, leading to a selective destruction of the chloroplast and the release of stromal constituents. Because 13-LOX pathway products comprise compounds involved in insect deterrence and pathogen defense (volatile aldehydes and oxylipins), a mechanism of unmolested nitrogen and carbon relocation is suggested that occurs from leaves to seeds and roots during fall.

It has been well established that DNA cytosine methylation plays essential regulatory roles in imprinting gene expression in endosperm, and hence normal embryonic development, in the model plant Arabidopsis (Arabidopsis thaliana). Nonetheless, the developmental role of this epigenetic marker in cereal crops remains largely unexplored. Here, we report for sorghum (Sorghum bicolor) differences in relative cytosine methylation levels and patterns at 5′-CCGG sites in seven tissues (endosperm, embryo, leaf, root, young inflorescence, anther, and ovary), and characterize a set of tissue-specific differentially methylated regions (TDMRs). We found that the most enriched TDMRs in sorghum are specific for the endosperm and are generated concomitantly but imbalanced by decrease versus increase in cytosine methylation at multiple 5′-CCGG sites across the genome. This leads to more extensive demethylation in the endosperm than in other tissues, where TDMRs are mainly tissue nonspecific rather than specific to a particular tissue. Accordingly, relative to endosperm, the other six tissues showed grossly similar levels though distinct patterns of cytosine methylation, presumably as a result of a similar extent of concomitant decrease versus increase in cytosine methylation that occurred at variable genomic loci. All four tested TDMRs were validated by bisulfite genomic sequencing. Diverse sequences were found to underlie the TDMRs, including those encoding various known-function or predicted proteins, transposable elements, and those bearing homology to putative imprinted genes in maize (Zea mays). We further found that the expression pattern of at least some genic TDMRs was correlated with its tissue-specific methylation state, implicating a developmental role of DNA methylation in regulating tissue-specific or -preferential gene expression in sorghum.

The potential of metchnikowin, a 26-amino acid residue proline-rich antimicrobial peptide synthesized in the fat body of Drosophila melanogaster was explored to engineer disease resistance in barley against devastating fungal plant pathogens. The synthetic peptide caused strong in vitro growth inhibition (IC(50) value approximately 1 muM) of the pathogenic fungus Fusarium graminearum. Transgenic barley expressing the metchnikowin gene in its 52-amino acid pre-pro-peptide form under the control of the inducible mannopine synthase (mas) gene promoter from the T(i) plasmid of Agrobacterium tumefaciens displayed enhanced resistance to powdery mildew as well as Fusarium head blight and root rot. In response to these pathogens, metchnikowin accumulated in plant apoplastic space, specifying that the insect signal peptide is functional in monocotyledons. In vitro and in vivo tests revealed that the peptide is markedly effective against fungal pathogens of the phylum Ascomycota but, clearly, less active against Basidiomycota fungi. Importantly, germination of the mutualistic basidiomycete mycorrhizal fungus Piriformospora indica was affected only at concentrations beyond 50 muM. These results suggest that antifungal peptides from insects are a valuable source for crop plant improvements and their differential activities toward different phyla of fungi denote a capacity for insect peptides to be used as selective measures on specific plant diseases.

In plant tissue culture, developmental disturbance and mutagenic factors are involved in channeling an individual totipotent cell to an intact plant. Comparing a pair of sorghum reciprocal F(1) hybrids with their parental pure lines revealed a dramatic difference in the occurrence of both genetic and DNA methylation alterations in the respective regenerated plants. In contrast to those of the pure lines, regenerated plants of hybrids exhibit significantly enhanced genetic and epigenetic stability. The genetic changes detected by amplified fragment length polymorphism and the DNA methylation alterations detected by methylation-sensitive amplified polymorphism are intimately correlated with each other, suggesting a common mechanism underlying both kinds of instabilities. Markedly altered transcription of genes encoding four putative sorghum DNA methyltransferases and two 5-methylcytosine glycosylases with nucleotide sequences orthologous to Arabidopsis counterparts was induced by tissue culture. The steady-state transcript levels of these genes were negatively correlated with genetic and methylation alterations. A salient observation is that tissue culture-induced transcription of genes encoding DNA methyltransferases and 5-methylcytosine glycosylases in calli and/or regenerated plants of the hybrids was remarkably coordinated, but is largely uncoordinated and stochastically altered in calli and/or regenerated plants of the pure lines. We suggest that the uncoordinated regulation of expression of DNA methyltransferases and 5-methylcytosine glycosylases is a major cause of the high incidence of genetic and DNA methylation alterations in cultures of pure lines, but coordinated up-regulated expression of these enzymes in cultures of the F(1) hybrids fortified their genetic and epigenetic stability.

Thioredoxins (Trxs) are ubiquitous small proteins with a redox-active disulfide bridge. In their reduced form, they constitute very efficient protein disulfide oxidoreductases. In chloroplasts, two types of Trxs ( f and m ) coexist and play central roles in the regulation of the Calvin cycle and other processes. Here, we identified a class of Trx targets in the inner plastid envelope membrane of chloroplasts that share a CxxC motif ≈73 aa from their carboxyl-terminal end. Members of this group belong to a superfamily of Rieske iron–sulfur proteins involved in protein translocation and chlorophyll metabolism. These proteins include the protein translocon protein TIC55, the precursor NADPH:protochlorophyllide oxidoreductase translocon protein PTC52, which operates as protochlorophyllide a -oxygenase, and the lethal leaf spot protein LLS1, which is identical with pheophorbide a oxygenase. The role of these proteins in dark/light regulation and oxidative control by the Trx system is discussed.

In plants, disease resistance mediated by the gene-for-gene mechanism involves the recognition of specific effector molecules produced by the pathogen either directly or indirectly by the resistance-gene products. This recognition triggers a series of signals, thereby serving as a molecular switch in regulating defense mechanisms by the plants. To understand the mechanism of action of the barley stem rust resistance gene Rpg1 , we investigated the fate of the RPG1 protein in response to infection with the stem rust fungus, Puccinia graminis f. sp. tritici . The investigations revealed that RPG1 disappears to undetectable limits only in the infected tissues in response to avirulent, but not virulent pathotypes. The RPG1 protein disappearance is rapid and appears to be due to specific protein degradation via the proteasome-mediated pathway as indicated by inhibition with the proteasomal inhibitor MG132, but not by other protease inhibitors.

Water-soluble chlorophyll proteins (WSCPs) constitute a small family of unusual chlorophyll (Chl)-binding proteins that possess a Kunitz-type protease inhibitor domain. In Arabidopsis thaliana, a WSCP has been identified, named AtWSCP, that forms complexes with Chl and the Chl precursor chlorophyllide (Chlide) in vitro. AtWSCP exhibits a quite unexpected expression pattern for a Chl binding protein and accumulated to high levels in the apical hook of etiolated plants. AtWSCP expression was negatively light-regulated. Transgenic expression of AtWSCP fused to green fluorescent protein (GFP) revealed that AtWSCP is localized to cell walls/apoplastic spaces. Biochemical assays identified AtWSCP as interacting with RD21 (RESPONSIVE TO DESICCATION 21), a granulin domain-containing cysteine protease implicated in stress responses and defense. Reconstitution experiments showed tight interactions between RD21 and WSCP that were relieved upon Chlide binding. Laboratory feeding experiments with two herbivorous isopod crustaceans, Porcellio scaber (woodlouse) and Armadillidium vulgare (pillbug), identified the apical hook as Achilles’ heel of etiolated plants and that this was protected by RD21 during greening. Because Chlide is formed in the apical hook during seedling emergence from the soil, our data suggest an unprecedented mechanism of herbivore resistance activation that is triggered by light and involves AtWSCP.