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Summary Aspergillus flavus and Fusarium verticillioides are fungal pathogens that colonize maize kernels and produce the harmful mycotoxins aflatoxin and fumonisin, respectively. Management practice based on potential host resistance to reduce contamination by these mycotoxins has proven difficult, resulting in the need for a better understanding of the infection process by these fungi and the response of maize seeds to infection. In this study, we followed the colonization of seeds by histological methods and the transcriptional changes of two maize defence-related genes in specific seed tissues by RNA in situ hybridization. Maize kernels were inoculated with either A. flavus or F. verticillioides 21–22 days after pollination, and harvested at 4, 12, 24, 48, 72, 96 and 120 h post-inoculation. The fungi colonized all tissues of maize seed, but differed in their interactions with aleurone and germ tissues. RNA in situ hybridization showed the induction of the maize pathogenesis-related protein, maize seed (PRms) gene in the aleurone and scutellum on infection by either fungus. Transcripts of the maize sucrose synthase-encoding gene, shrunken-1 (Sh1), were observed in the embryo of non-infected kernels, but were induced on infection by each fungus in the aleurone and scutellum. By comparing histological and RNA in situ hybridization results from adjacent serial sections, we found that the transcripts of these two genes accumulated in tissue prior to the arrival of the advancing pathogens in the seeds. A knowledge of the patterns of colonization and tissue-specific gene expression in response to these fungi will be helpful in the development of resistance.

Summary Aspergillus flavus is an opportunistic fungal pathogen that infects maize kernels pre-harvest, creating major human health concerns and causing substantial agricultural losses. Improved control strategies are needed, yet progress is hampered by the limited understanding of the mechanisms of infection. A series of studies were designed to investigate the localization, morphology and transcriptional profile of A. flavus during internal seed colonization. Results from these studies indicate that A. flavus is capable of infecting all tissues of the immature kernel by 96 h after infection. Mycelia were observed in and around the point of inoculation in the endosperm and were found growing down to the germ. At the endosperm–germ interface, hyphae appeared to differentiate and form a biofilm-like structure that surrounded the germ. The exact nature of this structure remains unclear, but is discussed. A custom-designed A. flavus Affymetrix GeneChip® microarray was used to monitor genome-wide transcription during pathogenicity. A total of 5061 genes were designated as being differentially expressed. Genes encoding secreted enzymes, transcription factors and secondary metabolite gene clusters were up-regulated and considered to be potential effector molecules responsible for disease in the kernel. Information gained from this study will aid in the development of strategies aimed at preventing or slowing down A. flavus colonization of the maize kernel.

Ribosome-inactivating proteins (RIPs, EC 3.2.2.22) are potent naturally occurring toxins found in numerous and diverse plant species. The maize RIP is unusual among the plant RIPs because it is synthesized as an inactive precursor (also known as maize proRIP1 or b-32). The proenzyme undergoes proteolytic activation that results in the removal of the NH(2)-terminal, the COOH-terminal, and internal sequences to form a two-chain holoenzyme capable of irreversibly modifying the large rRNA. The characterization of a second maize RIP (RIP2), encoded by the gene designated Rip3:2 is described here. Low levels of Rip3:2 RNA were detected in roots, shoots, tassels, silks, and leaves, but the Rip3:2 gene, unlike the Rip3:1 gene, is not under the control of the transcriptional activator Opaque-2. Instead, its expression was up-regulated by drought. Rip3:2 encodes a 31.1 kDa polypeptide that is very similar to proRIP1 in regions corresponding to those found in the active protein and the NH(2)-terminal extension. A 19-amino-acid internal portion of proRIP2 has little similarity to the proRIP1 sequence except that both are very rich in acidic residues. RIP activity assays revealed that Rip3:2 encodes a polypeptide that acquires RNA-specific N-glycosidase activity after proteolytic cleavage. Accumulation as inactive proenzymes may therefore be a general feature of maize RIPs. Differential regulation of the two RIP genes suggests that the corresponding proteins may be involved in defence-related functions with one being regulated developmentally and the other being responsive to an environmental stimulus.

The maize inbred Tex6 has resistance to colonization and aflatoxin accumulation by Aspergillus flavus. A protein inhibitory to growth of A. flavus has been identified from aqueous extracts of mature Tex6 seeds. This study reports the purification of a chitinase associated with this inhibitory activity to electrophoretic homogeneity and the further characterization of its properties. The inhibitory protein, which has an Mr of 29,000, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, is an endochitinase that is also capable of exochitinase activity. The enzyme has an optimal pH of 5.5 and a temperature optimum of 45°C. Chitinase activity in maize kernels peaked approximately 36 days after pollination. The Tex6 chitinase purified in this study is capable of inhibiting the growth of A. flavus by 50% at a concentration of 20 μg/ml. Our data indicate that chitinase activity in Tex6 kernels makes a major contribution to the antifungal activity in this maize genotype. Partial peptide sequence of the chitinase showed it to differ from previously reported chitinases.

Defective endosperm* (De*)-B30 is a dominant maize (Zea mays) mutation that depresses zein synthesis in the developing endosperm. The mutant kernels have an opaque, starchy phenotype, malformed zein protein bodies, and highly increased levels of binding protein and other chaperone proteins in the endosperm. Immunoblotting revealed a novel α-zein protein in De*-B30 that migrates between the 22- and 19-kD α-zein bands. Because the De*-B30 mutation maps in a cluster of 19-kD α-zein genes, we characterized cDNA clones encoding these proteins from a developing endosperm library. This led to the identification of a 19-kD α-zein cDNA in which proline replaces serine at the 15th position of the signal peptide. Although the corresponding gene does not appear to be highly expressed in De*-B30, it was found to be tightly linked with the mutant phenotype in a segregating F2 population. Furthermore, when the protein was synthesized in yeast cells, the signal peptide appeared to be less efficiently processed than when serine replaced proline. To test whether this gene is responsible for the De*-B30 mutation, transgenic maize plants expressing this sequence were created. T1 seeds originating from the transformants manifested an opaque kernel phenotype with enhanced levels of binding protein in the endosperm, similar to De*-B30. These results are consistent with the hypothesis that the De*-B30 mutation causes a defective signal peptide in a 19-kD α-zein protein.

Chitinases, beta-1,3-glucanases, and ribosome-inactivating proteins are reported to have antifungal activity in plants. With the aim of producing fungus-resistant transgenic plants, we co-expressed a modified maize ribosome-inactivating protein gene, MOD1, and a rice basic chitinase gene, RCH10, in transgenic rice plants. A construct containing MOD1 and RCH10 under the control of the rice rbcS and Act1 promoters, respectively, was co-transformed with a plasmid containing the herbicide-resistance gene bar as a selection marker into rice by particle bombardment. Several transformants analyzed by genomic Southern-blot hybridization demonstrated integration of multiple copies of the foreign gene into rice chromosomes. Immunoblot experiments showed that MOD1 formed approximately 0.5% of the total soluble protein in transgenic leaves. RCH10 expression was examined using the native polyacrylamide-overlay gel method, and high RCH10 activity was observed in leaf tissues where endogenous RCH10 is not expressed. R1 plants were analyzed in a similar way, and the Southern-blot patterns and levels of transgene expression remained the same as in the parental line. Analysis of the response of R2 plants to three fungal pathogens of rice, Rhizoctonia solani, Bipolaris oryzae, and Magnaporthe grisea, indicated statistically significant symptom reduction only in the case of R. solani (sheath blight). The increased resistance co-segregated with herbicide tolerance, reflecting a correlation between the resistance phenotype and transgene expression.

Maize kernels are susceptible to infection by the opportunistic pathogen Aspergillus flavus. Infection results in reduction of grain quality and contamination of kernels with the highly carcinogenic mycotoxin, aflatoxin. To understanding host response to infection by the fungus, transcription of approximately 9,000 maize genes were monitored during the host-pathogen interaction with a custom designed Affymetrix GeneChip® DNA array. More than 1,000 maize genes were found differentially expressed at a fold change of 2 or greater. This included the up regulation of defense related genes and signaling pathways. Transcriptional changes also were observed in primary metabolism genes. Starch biosynthetic genes were down regulated during infection, while genes encoding maize hydrolytic enzymes, presumably involved in the degradation of host reserves, were up regulated. These data indicate that infection of the maize kernel by A. flavus induced metabolic changes in the kernel, including the production of a defense response, as well as a disruption in kernel development.

The endoplasmic reticulum (ER) stress response is a signal transduction pathway activated by the perturbation of normal ER metabolism. We used the maize (Zea mays)floury-2 (fl2) mutant and soybean (Glycine max) suspension cultures treated with tunicamycin (Tm) to investigate the ER stress response as it relates to phospholipid metabolism in plants. Four key phospholipid biosynthetic enzymes, including DG kinase and phosphatidylinositol (PI) 4-phosphate 5-kinase were up-regulated in the fl2 mutant, specifically in protein body fractions where the mutation has its greatest effect. The third up-regulated enzyme, choline-phosphate cytidylyltransferase, was regulated by fl2 gene dosage and developmental signals. Elevated accumulation of the fourth enzyme, PI 4-kinase, was observed in the fl2 endosperm and soybean cells treated with Tm. The activation of these phospholipid biosynthetic enzymes was accompanied by alterations in membrane lipid synthesis and accumulation. The fl2 mutant exhibited increased PI content in protein body membranes at 18 d after pollination and more than 3-fold higher triacylglycerol accumulation in the endosperm by 36 d after pollination. Incorporation of radiolabeled acetate into phospholipids in soybean culture cells increased by about 30% with Tm treatment. The coordinated regulation of ER stress related proteins and multiple components of phospholipid biosynthesis is consistent with signaling through a common pathway. We postulate that the plant ER stress response has an important role in general plant metabolism, and more specifically in integrating the synthesis of protein and lipid reserves to allow proper seed formation.

The superfamily of ribosome-inactivating proteins (RIPs) consists of toxins that catalytically inactivate ribosomes at a universally conserved region of the large ribosomal RNA. RIPs carry out a single N-glycosidation event that alters the binding site of the translational elongational factor eEF1A and causes a cessation of protein synthesis that leads to subsequent cell death. Maize RIP1 is a kernel-specific RIP with the unusual property of being produced as a zymogen, proRIP1. ProRIP1 accumulates during seed development and becomes active during germination when cellular proteases remove acidic residues from a central domain and both termini. These deletions also result in RIP activation in vitro. However, the effectiveness of RIP1 activity against target ribosomes remains species-dependent. To determine the potential efficiency of maize RIP1 as a plant defense protein, we used quantitative RNA gel blots to detect products of RIP activity against intact ribosomal substrates from various species. We determined the enzyme specificity of recombinant maize proRIP1 (rproRIP1), papain-activated rproRIP1 and MOD1 (an active deletion mutant of rproRIP1) against ribosomal substrates with differing levels of RIP sensitivity. The rproRIP1 had no detectable enzymatic activity against ribosomes from any of the species assayed. The papain-activated rproRIP1 was more active than MOD1 against ribosomes from either rabbit or the corn pathogen, Aspergillus flavus, but the difference was much more marked when rabbit ribosomes were used as a substrate. The papain-activated rproRIP1 was much more active against rabbit ribosomes than homologous Zea mays ribosomes and had no detectable effect on Escherichia coli ribosomes.

We have previously transformed rice (Oyrza sativa L.) with the maize ribosome-inactivating protein b-32 gene (Zmcrip3a) and the phosphinothricin resistance gene (bar). In the present study, Southern blot hybridization analysis of 56 primary fertile transformants resulted in distinct band patterns, indicating that all the transformants had been generated by independent integration events and 30% of them contained a single copy of the transgene. Segregation analysis of 15 R0 plants revealed that transgene was stably transmitted to their progenies and Southern blot band patterns of R1 progenies remained the same as the corresponding parents, suggesting that all the loci of multiple integration events are genetically linked. Also, in most of the lines, physical presence of the b-32 transgene co-segregated with the phosphinothricin- resistant phenotype, confirming that the transgene is behaving as a normal locus in the genome. However, some of R1 seedlings that contained multiple copies of the transgene became sensitive to phosphinothricin, indicating that its expression was silenced. Immunoblot analysis demonstrated that b-32 protein was produced and the levels of expression differed in different lines, estimated to be 0.5–1% of total soluble protein in the leaf tissues. In addition, the transgene-encoded protein was preferentially processed in germinating seeds and young leaves of R2 transgenic plants in a way similar to that in maize kernels, suggesting that the processing mechanism is conserved in the germination stage between rice and maize.

The relative toxicity of proenzyme and protease-activated forms of maize seed ribosome-inactivating protein (b-32) to several insect species was determined. Only the protease-activated form had significant toxicity to any caterpillars when fed in diets at 1 mg/g of diet. Activity ranged from 70% mortality to cabbage looper (Trichoplusia ni) to no effect to Indian meal moth (Plodia interpunctella). Neither form of the protein showed activity against larvae of the Freeman sap beetle, (Carpophilus freemani). However, the proenzyme and protease-activated forms were approximately equally deterrent in choice assays to other sap beetles and maize weevils (Sitophilus zeamais), with relative feeding rates reduced by up to 6-fold. Because this protein can naturally occur at the 1 mg/g endosperm range in the endosperm of Opaque-2 (normal) plants versus 2 orders of magnitude lower in opaque-2 mutants, it is likely that this RIP plays a natural defensive role against insects. However, some insects appear to have adapted to this protein.

The maize (Zea mays L.) floury-2 (fl2) mutation is associated with a general decrease in storage protein synthesis, altered protein body morphology, and the synthesis of a novel 24-kD α--zein storage protein. Unlike storage proteins in normal kernels and the majority of storage proteins in fl2 kernels, the 24-kD α--zein contains a signal peptide that would normally be removed during protein synthesis and processing. The expected processing site of this α--zein reveals a putative mutation alaine->valine (Ala->Val) that is not found at other junctions between signal sequences and mature proteins. To investigate the impact of such a mutation on signal peptide cleavage, we have assayed the 24-kD fl2 α--zein in a co-translational processing system in vitro. Translation of RNA from fl2 kernels or synthetic RNA encoding the fl2 α--zein in the presence of microsomes yielded a 24-kD polypeptide. A normal signal peptide sequence, generated by site-directed mutagenesis, restored the capacity of the RNA to direct synthesis of a properly processed protein in a cell-free system. Both the fl2 α--zein and the fl2 α--zein (Val->Ala) were translocated into the lumen of the endoplasmic reticulum. The processed fl2 α--zein (Val->Ala) was localized in the soluble portion of the microsomes, whereas the fl2 α--zein co-fractionated with the microsomal membranes. By remaining anchored to protein body membranes during endosperm maturation, the fl2 zein may thus constrain storage protein packing and perturb protein body morphology.

The ER luminal binding protein, BiP, has been linked to prolamine protein body formation in rice. To obtain further information on the possible role of this chaperone in protein body formation we have cloned and sequenced a BiP cDNA homolog from rice endosperm. The rice sequence is very similar to the maize BiP exhibiting 92% nucleotide identity and 96% deduced amino acid sequence identity in the coding region. Substantial amino acid sequence homology exists between rice BiP and BiP homologs from several other plant and animal species including long stretches of conservation through the amino-terminal ATPase domain. Considerable variation, however, is observed within the putative carboxy-terminal peptide-binding domain between the plant and nonplant BiP sequences. A single hand of approximately 2.4 kb was visible when RNA gel blots of total RNA purified from seed tissue were probed with radiolabeled rice BiP cDNA. This band increased in intensity during seed development up to 10 days after flowering, and then decreased gradually until seed maturity. Protein gel blots indicated that BiP polypeptide accumulation parallels that of the prolamine polypeptides throughout seed development. Immunocytochemical analysis demonstrated that BiP is localized in a non-stochastic fashion in the endoplasmic reticulum membrane complex of developing endosperm cells. It is abundant on the periphery of the protein inclusion body but not in the central portion of the protein body or in the cisternal ER membranes connecting the protein bodies. These data support a model which proposes that BiP associates with the newly synthesized prolamine polypeptide to facilitate its folding and assembly into a protein inclusion body, and is then recycled.

Aspergillus flavus is an opportunistic fungal pathogen that infects maize kernels pre‐harvest, creating major human health concerns and causing substantial agricultural losses. Improved control strategies are needed, yet progress is hampered by the limited understanding of the mechanisms of infection. A series of studies were designed to investigate the localization, morphology and transcriptional profile of A. flavus during internal seed colonization. Results from these studies indicate that A. flavus is capable of infecting all tissues of the immature kernel by 96 h after infection. Mycelia were observed in and around the point of inoculation in the endosperm and were found growing down to the germ. At the endosperm–germ interface, hyphae appeared to differentiate and form a biofilm‐like structure that surrounded the germ. The exact nature of this structure remains unclear, but is discussed. A custom‐designed A. flavus Affymetrix GeneChip® microarray was used to monitor genome‐wide transcription during pathogenicity. A total of 5061 genes were designated as being differentially expressed. Genes encoding secreted enzymes, transcription factors and secondary metabolite gene clusters were up‐regulated and considered to be potential effector molecules responsible for disease in the kernel. Information gained from this study will aid in the development of strategies aimed at preventing or slowing down A. flavus colonization of the maize kernel.

The maize RIP1 gene encodes proRIP, the zymogen form of a ribosome-inactivating protein that accumulates to high levels in endosperm. ProRIP is synthesized coordinately with the major storage proteins and requires a functional allele of the transcriptional activator, Opaque-2 (O2)3 for maximal accumulation. RIP1 gene expression was characterized by developmental RNA gel blot analysis. RIP1 RNA was first detected at 10 days after pollination (DAP), and increased in abundance to reach maximal levels at mid-maturation of the kernel. In four W64A maize lines with independently-derived opaque-2 (o2) alleles, RIP1 RNA still accumulated in a developmentally specific pattern, but showed qualitative and quantitative changes from normal. RNA gel blots reprobed with an O2-specific probe provided evidence for coordinate accumulation of RIP1 and O2 RNAs throughout normal kernel development. The pleiotropic endosperm mutations brittle-1 (bt1), brittle-2 (bt2), shrunken-2 (sh2), sugary-2 (su2), Mucronate (Mc), Defective endosperm-B30 (De∗-B30), and floury-2 (fl2), affected RIP1 and O2 RNA accumulation to varying degrees. None of these mutations, however, resulted in RIP1 RNA reductions comparable to that seen in W64Ao2. When coupled with o2, bt1 and bt2 mutations showed synergistic interactions in their effects of reducing RIP1 RNA levels. Thus, RIP1 gene expression and relative RNA levels are controlled by multiple factors including O2, and o2-independent component(s), genetic background, and the presence of other pleiotropic mutations.

A maize 75 kDa protein recently has been identified as a plant homolog of the mammalian binding protein (BiP). To better understand the function of BiP in protein body formation in maize endosperm, immunomicroscopy studies were conducted on three maize endosperm mutants, floury-2, Mucronate, and Defective endosperm-B 30, in which the level of BiP is highly elevated. Our results showed that protein body morphology in all three mutants was altered. In addition, BiP was localized in both the ER and peripheral regions of the abnormal protein bodies. The degree to which protein body morphology differed from normal was positively correlated with increased amounts of BiP. In addition, the accumulation of BiP in abnormal protein bodies increased with protein body maturation. In the three endosperm mutants, the arrangement of zeins within protein bodies had been perturbed, yet none of the specific zein subclasses exhibited the staining pattern found for BiP. The association of BiP with abnormal packaging of proteins in protein bodies may reflect a biological function to mediate protein folding and assembly in maize endosperm.

Zeins, the most abundant proteins in the maize seed, have been studied for over 100 years. We have learned a great deal about the structure, synthesis and genetic regulation of these proteins, making them a valuable model system to study storage protein synthesis. In this review, we explore longstanding questions and controversies regarding zeins in light of new information from genomic sequencing, improved protein detection methods and molecular characterization of mutants.

The plant hormone indole-3-acetic acid (IAA) is the most abundant natural auxin involved in many aspects of plant development and growth. The IAA levels in plants are modulated by a specific group of amidohydrolases from the peptidase M20D family that release the active hormone from its conjugated storage forms. Here, we describe the X-ray crystal structure of IAA-amino acid hydrolase IAA-leucine resistantlike gene 2 (ILL2) from Arabidopsis thaliana at 2.0 A resolution. ILL2 preferentially hydrolyses the auxin-amino acid conjugate N-(indol-3-acetyl)-alanine. The overall structure of ILL2 is reminiscent of dinuclear metallopeptidases from the M20 peptidase family. The structure consists of two domains, a larger catalytic domain with three-layer alpha beta alpha sandwich architecture and aminopeptidase topology and a smaller satellite domain with two-layer alphabeta-sandwich architecture and alpha-beta-plaits topology. The metal-coordinating residues in the active site of ILL2 include a conserved cysteine that clearly distinguishes this protein from previously structurally characterized members of the M20 peptidase family. Modeling of N-(indol-3-acetyl)-alanine into the active site of ILL2 suggests that Leu175 serves as a key determinant for the amino acid side-chain specificity of this enzyme. Furthermore, a hydrophobic pocket nearby the catalytic dimetal center likely recognizes the indolyl moiety of the substrate. Finally, the active site of ILL2 harbors an absolutely conserved glutamate (Glu172), which is well positioned to act as a general acid-base residue. Overall, the structure of ILL2 suggests that this enzyme likely uses a catalytic mechanism that follows the paradigm established for the other enzymes of the M20 peptidase family.

NRPs (N-rich proteins) were identified as targets of a novel adaptive pathway that integrates endoplasmic reticulum (ER) and osmotic stress signals based on coordinate regulation and synergistic up-regulation by tunicamycin and polyethylene gly- col treatments. This integrated pathway diverges from the molecular chaperone-inducing branch of the unfolded protein response (UPR) in several ways. While UPR-specific targets were inversely regulated by ER and osmotic stresses, NRPs required both signals for full activation. Furthermore, BiP (binding protein) overexpression in soybean prevented activa- tion of the UPR by ER stress inducers, but did not affect activa- tion of NRPs. We also found that this integrated pathway trans- duces a PCD signal generated by ER and osmotic stresses that result in the appearance of markers associated with leaf senes- cence. Overexpression of NRPs in soybean protoplasts induced caspase-3-like activity and promoted extensive DNA fragmen- tation. Furthermore, transient expression of NRPs in planta caused leaf yellowing, chlorophyll loss, malondialdehyde pro- duction, ethylene evolution, and induction of the senescence marker gene CP1. This phenotype was alleviated by the cytoki- nin zeatin, a potent senescence inhibitor. Collectively, these results indicate that ER stress induces leaf senescence through activation of plant-specific NRPs via a novel branch of the ER stress response.

Three DNA regions required for high levels of transcription were identified by transient gene expression analysis of the 5' flanking region of a 19 kDa alpha-zein gene. For these analyses, the zein promoter region was fused to the beta-glucuronidase (GUS) gene and assayed by transient expression in carrot protoplasts. A 107-bp sequence (-114/-8) containing the TATA box resulted in low levels of GUS activity. Addition of the proximal 75 bp (-189/-114) doubled the level of GUS expression, and a further increase in expression was obtained when additional upstream sequences (-483/-226) were placed 5' of the zein promoters. Zein upstream sequences enhanced transcription independently of the -189/-114 region. Although the -189/-114 region was not essential for transcription, it was important to obtain maximum GUS activity. A 121 bp upstream sequence (-347/-226) that contains the conserved TGTAAAG sequence gave high levels of GUS activity when placed in either orientation 5' of the zein promoter sequences. However, nucleotides -347 to -309, containing the TGTAAAG sequence, could be deleted from this fragment without a significant change in GUS activity. Zein upstream sequences did not promote transcription of the GUS gene in somatic maize protoplasts. The upstream activating sequence from the cauliflower mosaic virus (CaMV) 35S promoter placed 5' of deletion mutants of the zein promoter also failed to produce GUS activity above background.

Background Despite the potential of the endoplasmic reticulum (ER) stress response to accommodate adaptive pathways, its integration with other environmental-induced responses is poorly understood in plants. We have previously demonstrated that the ER-stress sensor binding protein (BiP) from soybean exhibits an unusual response to drought. The members of the soybean BiP gene family are differentially regulated by osmotic stress and soybean BiP confers tolerance to drought. While these results may reflect crosstalk between the osmotic and ER-stress signaling pathways, the lack of mutants, transcriptional response profiles to stresses and genome sequence information of this relevant crop has limited our attempts to identify integrated networks between osmotic and ER stress-induced adaptive responses. As a fundamental step towards this goal, we performed global expression profiling on soybean leaves exposed to polyethylene glycol treatment (osmotic stress) or to ER stress inducers. Results The up-regulated stress-specific changes unmasked the major branches of the ER-stress response, which include enhancing protein folding and degradation in the ER, as well as specific osmotically regulated changes linked to cellular responses induced by dehydration. However, a small proportion (5.5%) of total up-regulated genes represented a shared response that seemed to integrate the two signaling pathways. These co-regulated genes were considered downstream targets based on similar induction kinetics and a synergistic response to the combination of osmotic- and ER-stress-inducing treatments. Genes in this integrated pathway with the strongest synergistic induction encoded proteins with diverse roles, such as plant-specific development and cell death (DCD) domain-containing proteins, an ubiquitin-associated (UBA) protein homolog and NAC domain-containing proteins. This integrated pathway diverged further from characterized specific branches of ER-stress as downstream targets were inversely regulated by osmotic stress. Conclusion The present ER-stress- and osmotic-stress-induced transcriptional studies demonstrate a clear predominance of stimulus-specific positive changes over shared responses on soybean leaves. This scenario indicates that polyethylene glycol (PEG)-induced cellular dehydration and ER stress elicited very different up-regulated responses within a 10-h stress treatment regime. In addition to identifying ER-stress and osmotic-stress-specific responses in soybean (Glycine max), our global expression-profiling analyses provided a list of candidate regulatory components, which may integrate the osmotic-stress and ER-stress signaling pathways in plants.

Pre-harvest and post-harvest contamination of maize, peanuts, cotton, and tree nuts by members of the genus Aspergillus and subsequent contamination with the mycotoxin aflatoxin pose a widespread food safety problem for which effective and inexpensive control strategies are lacking. Since the discovery of aflatoxin as a potently carcinogenic food contaminant, extensive research has been focused on identifying compounds that inhibit its biosynthesis. Numerous diverse compounds and extracts containing activity inhibitory to aflatoxin biosynthesis have been reported. Only recently, however, have tools been available to investigate the molecular mechanisms by which these inhibitors affect aflatoxin biosynthesis. Many inhibitors are plant-derived and a few may be amenable to pathway engineering for tissue-specific expression in susceptible host plants as a defense against aflatoxin contamination. Other compounds show promise as protectants during crop storage. Finally, inhibitors with different modes of action could be used in comparative transcriptional and metabolomic profiling experiments to identify regulatory networks controlling aflatoxin biosynthesis.

Mucronate (Mc) was identified as a dominant maize (Zea mays L.) opaque kernel mutation that alters zein storage protein synthesis. Zein protein bodies in Mc endosperm are misshapen and are associated with increased levels of ER Lumenal Binding Protein (BiP). Using GeneCalling to profile endosperm RNA transcripts, we identified an aberrant RNA in Mc that encodes the 16-kDa gamma-zein protein. The transcript contains a 38-bp deletion (nucleotides 406-444 after the initiation codon) that creates a frame-shift mutation and an abnormal sequence for the last 63 amino acids. Genetic mapping revealed the Mc mutation is linked with the locus encoding the 16-kDa gamma-zein, and two-dimensional gel electrophoresis confirmed the 16-kDa gamma-zein protein is altered in Mc. The mutant protein exhibited changes in solubility properties and co-immunoprecipitated with the molecular chaperone, BiP. Transgenic maize plants expressing the Mc 16-kDa gamma-zein manifested an opaque kernel phenotype with enhanced levels of BiP in the endosperm, similar to the Mc mutant. Unlike the wild-type protein, the Mc 16-kDa gamma-zein interacted only weakly with the 22-kDa alpha-zein when expressed in the yeast two-hybrid system. These results indicate that the Mc phenotype results from a frame-shift mutation in the gene encoding the 16-kDa gamma-zein protein, leading to the unfolded protein response in developing endosperm.

Tobacco (Nicotiana tabacum) plants grown from seed obtained by crossing a tobacco line that expressed an activated maize ribosome-inactivating protein (RIP) with a line that overexpressed tobacco anionic peroxidase were tested for their effects on corn earworm Helicoverpa zea and cigarette beetle Lasioderma serricorne larvae as compared to the wild-type plant cross. Significant feeding reductions were noted for transgenic plants expressing both resistance proteins as compared to wild-type plants for both H. zea and L. serricorne. Significant increases in mortality were also noted for those insects fed on the transgenic cross as compared to wild-type plants in some cases. Levels of both peroxidase and maize RIP were significantly higher in transgenic as compared to wild-type plants (which did not produce maize RIP). The degree of feeding was significantly negatively correlated with the level of RIP or peroxidase individually. Keywords: Helicoverpa; Lasioderma; peroxidase; ribosome-inactivating protein; ins...

The disposal of misfolded proteins from the lumen of the endoplasmic reticulum (ER) is one of the quality control mechanisms present in the protein secretory pathway. Through ER-associated degradation, misfolded substrates are targeted to the cytosol where they are degraded by the proteasome. We have identified four maize (Zea mays) Der1-like genes (Zm Derlins) that encode homologs of Der1p, a yeast (Saccharomyces cerevisiae) protein implicated in ER-associated degradation. Zm Derlins are capable of functionally complementing a yeast Der1 deletion mutant. Such complementation indicates that the Der1p function is conserved among species. Zm Derlin genes are expressed at low levels throughout the plant, but appear prevalent in tissues with high activity of secretory protein accumulation, including developing endosperm cells. Expression of three of the four Zm Derlin genes increases during ER stress, with Zm Derlin1-1 showing the strongest induction. Subcellular fractionation experiments localized Zm Derlin proteins to the membrane fraction of microsomes. In maize endosperm, Zm Derlin proteins were found primarily associated with ER-derived protein bodies regardless of the presence of an ER stress response.

Protein disulfide isomerases (PDIs) are molecular chaperones that contain thioredoxin (TRX) domains and aid in the formation of proper disulfide bonds during protein folding. To identify plant PDI-like (PDIL) proteins, a genome-wide search of Arabidopsis (Arabidopsis thaliana) was carried out to produce a comprehensive list of 104 genes encoding proteins with TRX domains. Phylogenetic analysis was conducted for these sequences using Bayesian and maximum-likelihood methods. The resulting phylogenetic tree showed that evolutionary relationships of TRX domains alone were correlated with conserved enzymatic activities. From this tree, we identified a set of 22 PDIL proteins that constitute a well-supported clade containing orthologs of known PDIs. Using the Arabidopsis PDIL sequences in iterative BLAST searches of public and proprietary sequence databases, we further identified orthologous sets of 19 PDIL sequences in rice (Oryza sativa) and 22 PDIL sequences in maize (Zea mays), and resolved the PDIL phylogeny into 10 groups. Five groups (I–V) had two TRX domains and showed structural similarities to the PDIL proteins in other higher eukaryotes. The remaining five groups had a single TRX domain. Two of these (quiescin-sulfhydryl oxidase-like and adenosine 5′-phosphosulfate reductase-like) had putative nonisomerase enzymatic activities encoded by an additional domain. Two others (VI and VIII) resembled small single-domain PDIs from Giardia lamblia, a basal eukaryote, and from yeast. Mining of maize expressed sequence tag and RNA-profiling databases indicated that members of all of the single-domain PDIL groups were expressed throughout the plant. The group VI maize PDIL ZmPDIL5-1 accumulated during endoplasmic reticulum stress but was not found within the intracellular membrane fractions and may represent a new member of the molecular chaperone complement in the cell.

Chitinases, beta-1,3-glucanases, and ribosome-inactivating proteins are reported to have antifungal activity in plants. With the aim of producing fungus-resistant transgenic plants, we co-expressed a modified maize ribosome-inactivating protein gene, MOD1, and a rice basic chitinase gene, RCH10, in transgenic rice plants. A construct containing MOD1 and RCH10 under the control of the rice rbcS and Act1 promoters, respectively, was co-transformed with a plasmid containing the herbicide-resistance gene bar as a selection marker into rice by particle bombardment. Several transformants analyzed by genomic Southern-blot hybridization demonstrated integration of multiple copies of the foreign gene into rice chromosomes. Immunoblot experiments showed that MOD1 formed approximately 0.5% of the total soluble protein in transgenic leaves. RCH10 expression was examined using the native polyacrylamide-overlay gel method, and high RCH10 activity was observed in leaf tissues where endogenous RCH10 is not expressed. R1 plants were analyzed in a similar way, and the Southern-blot patterns and levels of transgene expression remained the same as in the parental line. Analysis of the response of R2 plants to three fungal pathogens of rice, Rhizoctonia solani, Bipolaris oryzae, and Magnaporthe grisea, indicated statistically significant symptom reduction only in the case of R. solani (sheath blight). The increased resistance co-segregated with herbicide tolerance, reflecting a correlation between the resistance phenotype and transgene expression.

Progeny of two transgenic tobacco (Nicotiana tabacum L.) lines that expressed an activated form of maize (Zea mays L.) ribosome-inactivating protein (RIP) had varying resistance to the insect species tested. A subset of R(2) plants from the two lines appeared to be more resistant to larvae of the cigarette beetle, Lasioderma serricorne (F.), and the tobacco hornworm, Manduca sexta (L.) than the wild type plants. Progeny (R(3)) of the more resistant R(2) plants were tested more extensively for insect resistance. Resistance to the corn earworm, Helicoverpa zea (Boddie), was most consistent, with significantly decreased feeding often accompanied by increased mortality and reduced weights of survivors fed on leaf disks of the two transgenic lines compared to the wild type. The amount of damage by H. zea was significantly inversely correlated with levels of RIP. Resistance of RIP-producing plants to H. zea was greater than expected on the basis of prior in vitro results using diet-incorporated maize RIP. The R(3) transgenic plant leaf disks were also often more resistant to feeding by larvae of L. serricorne compared to wild type plants. Although reduced feeding by M. sexta was noted when they were fed leaf disks from transgenic compared to wild type plants the first day of exposure, differences were not significant. This information provides further support for maize RIP having a role in resistance to maize-feeding insects.

▪ Abstract Ribosome-inactivating proteins (RIPs) are toxic N-glycosidases that depurinate the universally conserved α-sarcin loop of large rRNAs. This depurination inactivates the ribosome, thereby blocking its further participation in protein synthesis. RIPs are widely distributed among different plant genera and within a variety of different tissues. Recent work has shown that enzymatic activity of at least some RIPs is not limited to site-specific action on the large rRNAs of ribosomes but extends to depurination and even nucleic acid scission of other targets. Characterization of the physiological effects of RIPs on mammalian cells has implicated apoptotic pathways. For plants, RIPs have been linked to defense by antiviral, antifungal, and insecticidal properties demonstrated in vitro and in transgenic plants. How these effects are brought about, however, remains unresolved. At the least, these results, together with others summarized here, point to a complex biological role. With genetic, genomic, molecular, and structural tools now available for integrating different experimental approaches, we should further our understanding of these multifunctional proteins and their physiological functions in plants.

Using pulse–chase experiments combined with immunoprecipitation and N-glycan structural analysis, we showed that the retrieval mechanism of proteins from post–endoplasmic reticulum (post-ER) compartments is active in plant cells at levels similar to those described previously for animal cells. For instance, recycling from the Golgi apparatus back to the ER is sufficient to block the secretion of as much as 90% of an extracellular protein such as the cell wall invertase fused with an HDEL C-terminal tetrapeptide. Likewise, recycling can sustain fast retrograde transport of Golgi enzymes into the ER in the presence of brefeldin A. However, on the basis of our data, we propose that this retrieval mechanism in plants has little impact on the ER retention of a soluble ER protein such as calreticulin. Indeed, the latter is retained in the ER without any N-glycan–related evidence for a recycling through the Golgi apparatus. Taken together, these results indicate that calreticulin and perhaps other plant reticuloplasmins are possibly largely excluded from vesicles exported from the ER. Instead, they are probably retained in the ER by mechanisms that rely primarily on signals other than H/KDEL motifs.

Binding protein (BiP) is the endoplasmic reticulum member of the highly conserved HSP70 (heat shock protein 70) family of molecular chaperones. We have isolated and characterized two different BiP cDNA clones corresponding to genes expressed in immature kernels. These two cDNAs share extensive sequence similarity but map to unlinked loci in the maize genome. A comparison of the aa sequences predicted from the cDNA clones revealed only six aa differences between them. Investigation of gene-specifc expression was carried out by RNA gel blot analysis. RNAs corresponding to both cDNA clones were present in increased amounts in the endosperm of floury-2 ( fl2 ), Mucronate (Mc) and Defective endosperm-B30 (De * -B30) maize mutants, which produce abnormal storage proteins. Similar increases in RNAs corresponding to both probes were detected in cells treated with either of two agents that interfere with protein folding, azetidine-2-carboxylic acid (AZC) and tunicamycin. Investigation of the genomic complexity of the BiP genes by Southern blot analysis revealed several cross-hybridizing bands. These results are suggestive that the BiP genes expressed in endosperm are coordinately regulated members of a more complex maize BiP multigene family.

The maize floury 2 (fl2) mutation enhances the lysine content of the grain, but the soft texture of the endosperm makes it unsuitable for commercial production. The mutant phenotype is linked with the appearance of a 24-kDa alpha-zein protein and increased synthesis of binding protein, both of which are associated with irregularly shaped protein bodies. We have cloned the gene encoding the 24-kDa protein and show that it is expressed as a 22-kDa alpha-zein with an uncleaved signal peptide. Comparison of the deduced N-terminal amino acid sequence of the 24-kDa alpha-zein protein with other alpha-zeins revealed an alanine to valine substitution at the C-terminal position of the signal peptide, a histidine insertion within the seventh alpha-helical repeat, and an alanine to threonine substitution with the same alpha-helical repeat of the protein. Structural defects associated with this alpha-zein explain many of the phenotypic effects of the fl2 mutation.

Publisher Summary This chapter discusses protein–protein interactions within the endoplasmic reticulum. The translocation of proteins into the endoplasmic reticulum (ER) is the initial transport step for proteins with ER, Golgi, lysosomal, vacuolar, or extracellular destinations. The process of protein translocation into the ER involves a complex series of events and interactions among numerous proteins, including the nascent polypeptide, signal recognition particle, signal-sequence receptor, signal peptidase, and several ER-resident proteins. Recent work in yeast, animal, and plant systems has implicated ER-resident proteins in the folding, assembly, and modification of proteins translocated into the ER. Most seed storage proteins are transported through the ER en route to specialized storage organelles. This chapter focuses on the identification of protein–protein interactions within the ER and ER-derived protein bodies. Successful detection of the interaction between two polypeptides requires a relatively stable protein–protein complex, steric availability of recognizable epitopes upon protein association, and no cross-reactivity between antibodies and associated proteins.

Rice prolamines are sequestered within the endoplasmic reticulum (ER) lumen even though they lack a lumenal retention signal. Immunochemical and biochemical data show that BiP, a protein that binds lumenal polypeptides, is localized on the surface of the aggregated prolamine protein bodies (PBs). BiP also forms complexes with nascent chains of prolamines in polyribosomes and with free prolamines with distinct adenosine triphosphate sensitivities. Thus, BiP retains prolamines in the lumen by facilitating their folding and assembly into PBs.

Although synthesis of the cytosolic maize albumin b-32 had been shown to be controlled by the Opaque-2 regulatory locus, its function was unknown. We show here that b-32 is a member of the large and widely distributed class of toxic plant proteins with ribosome-inactivating activity. These ribosome-inactivating proteins (RIPs) are RNA N-glycosidases that remove a single base from a conserved 28S rRNA loop required for elongation factor 1 alpha binding. Cell-free in vitro translation extracts were used to show that both maize and wheat ribosomes were resistant to molar excesses of b-32 but not to the dicotyledonous RIP gelonin. We extracted RIP activity from kernels during seed maturation and germination. The amount of RIP activity increased during germination, although the amount of b-32 protein remained fairly constant. Expression of a maize RIP gene under the control of an endosperm-specific transcriptional regulatory may be an important clue prompting investigation of the biological basis for RIP expression in seeds of other plants.

Plants carrying floury-2, Defective endosperm-B30, or Mucronate mutations overproduce b-70, a maize homolog of the mammalian immunoglobulin binding protein. During endosperm development in these mutants, levels of both b-70 protein and RNA increase dramatically between 14 days and 20 days after pollination. At later stages, b-70 RNA levels decline while protein levels remain high. The increase in b-70 RNA levels is endosperm specific and dependent on gene dosage in the floury-2 mutant. In all three mutants, the increases in b-70 RNA and protein levels are inversely proportional to changes in zein synthesis. Although b-70 polypeptides can be extracted from purified protein bodies, they carry a carboxy-terminal endoplasmic reticulum retention signal, HDEL. We propose that induction of b-70 in these mutants is a cellular response to abnormally folded or improperly assembled storage proteins and probably reflects its role as a polypeptide chain binding protein.

The maize b-70 protein is an endoplasmic reticulum protein overproduced in the floury-2 (fl2) endosperm mutant. The increase in b-70 levels in fl2 plants occurs during seed maturation and is endosperm specific. We have used amino acid sequence homology to identify b-70 as a homolog of mammalian immunoglobulin binding protein (BiP). Purified b-70 fractions contain two 75-kilodalton polypeptides with pl values of 5.3 and 5.4. Both 75-kilodalton polypeptides share several properties with BiP, including the ability to bind ATP and localization within the lumen of the endoplasmic reticulum. In addition, both b-70 polypeptides can be induced in maize cell cultures with tunicamycin treatment. Like BiP, the pl 5.3 form of b-70 is post-translationally modified by phosphorylation and ADP-ribosylation. However, modification of the pl 5.4 species was not detected in vitro or in vivo. Although the b-70 gene is unlinked to fl2, b-70 overproduction is positively correlated with the fl2 gene and is regulated at the mRNA level. In contrast, the fl2 allele negatively affects the accumulation of the major endosperm storage proteins. The physical similarity of b-70 to BiP and its association with abnormal protein accumulation in fl2 endoplasmic reticulum may reflect a biological function to mediate protein folding and assembly in maize endosperm.

Wheat (Triticum aestivum) RNA was used to program synthesis of the alpha-amylase protein by Xenopus laevis oocytes. A 41,500-dalton protein was made which was identified as alpha-amylase by immunoprecipitation with rabbit anti-alpha-amylase antiserum raised against the purified wheat protein and by its co-migration with authentic alpha-amylase on sodium dodecyl sulfate polyacrylamide gels. Synthesis of alpha-amylase was dependent upon injection of RNA extracted from gibberellic acid-induced aleurone layers from wheat. The amount of alpha-amylase produced was proportional to the amount of RNA injected and reached a plateau within 4 hours after injection. When the same RNA was translated in a wheat germ cell-free translation system, a 43,000-dalton protein was produced. Addition of dog pancreas microsomal membranes to the wheat germ translation system resulted in processing of the alpha-amylase protein to a form which co-migrated with authentic alpha-amylase purified from malted wheat and with the protein synthesized in oocytes.

Plasmids were constructed that contained promoters of `plant9 genes fused to the bacterial gene for chloramphenicol acetyl transferase. The promoters were isolated from a developmentally regulated Zea mays seed storage protein gene and from the mannopine synthase gene of the octopine type Ti plasmid of Agrobacterium tumefaciens which is constitutively expressed in crown gall tumors. These plasmids were introduced into carrot protoplasts made permeable by electroporation. Expression of chloramphenicol acetyl transferase activity directed by both promoters was positively correlated with DNA concentration. The efficiency of gene transfer was increased by inclusion of polyethylene glycol and by optimization of the voltage used in electroporation.

A maize genomic clone containing a 15 kilodalton zein gene was used as a template in an in vitro transcription system for HeLa cells. A runoff assay indicated transcription was initiating 5' to the map position of the open reading frame for the protein. Fine-structure mapping of RNAs synthesized in vitro showed two transcription start sites separated by 24 bases. One start site is 27 bases downstream of a consensus TATA sequence; the other is 30 bases downstream of a TATG sequences. The initiation sites for RNA synthesized in vitro map to the same region of the genomic clone as zein RNA isolated from developing maize kernels.

Seed storage proteins provide useful model systems for the study of gene regulation. These proteins are found only in the seed and in most angiosperms account for approximately half of the total seed protein. The storage proteins in maize seed are a group of prolamin proteins called zeins. Zeins are synthesized in the developing endosperm by membrane-bound polyribosomes and associate into insoluble aggregates called “protein bodies” within the lumen of the rough endoplasmic reticulum (1). Zein proteins are isolated from either mature seed or protein bodies of developing seeds by extraction with alcoholic solutions (70% ethanol or 55% isopropanol) in the presence of reducing agents such as 2-mercaptoethanol. Extraction with 70% alcohol alone results in recovery of proteins of 22 and 19 kD while extraction with alcohol plus 2-mercaptoethanol yields proteins of 27, 22, 19, 15, 14, and 10 kD (2). When separated by 2-dimensional gel electrophoresis, the 22 and 19 kD components each resolve into a number of charged isomers, but the proteins of 27, 15, 14, and 10 kD migrate as single polypeptides. Based on immunocytochemical analysis, the 22 and 19 kD proteins are distributed throughout the matrix of the protein body, whereas the 27, 15, and 14 kD proteins are localized near its surface (C. Lending, A. L. Kriz, C. Bracker and B. A. Larkins, unpublished data).

Analysis of a series of clones containing deletions in the 5' noncoding sequence of a gene encoding an Mr 19,000 zein allowed identification of a region required for maximal transcription. Transcriptional activity was assayed in two heterologous plant systems. In one system, the Ti plasmid was used to introduce the modified zein genes into the sunflower genome. In the other system, electroporation was used to transform carrot protoplasts with plasmids containing the zein genes. For the electroporation experiments, the 5' noncoding sequences from the zein clones were linked to the protein coding sequence of chloramphenicol acetyl transferase. The results showed that an upstream sequence, delimited by nucleotides -337 and -125 with respect to the mRNA cap site, is required for maximal transcription of the gene. In contrast, very low levels of transcription were directed by constructs that contained 125 bp of 5' noncoding sequence that included the CAAT and TATA boxes, suggesting that the additional sequences (-337 to -125) further 5' exert a quantitative effect on transcription. Examination of the additional 5' sequences showed five regions that share homology with the SV40 enhancer core sequence.

Zeins, the storage proteins of maize seed, are encoded by a large multigene family that is regulated developmentally and expressed in a tissue-specific manner during endosperm development. The synthesis of these proteins is affected by mutations, such as opaque-2, that cause a reduction in the accumulation of zein proteins and mRNAs. We used nuclear run-on transcription assays to analyze the expression of zein genes in developing normal and opaque-2 endosperms and to map the origin of these transcripts with respect to the coding and noncoding regions of the genes. These analyses demonstrate that zein gene expression is regulated transcriptionally and posttranscriptionally in developing endosperm. Transcription of genes encoding alpha-zeins is inhibited significantly in opaque-2 mutants, with expression of those encoding the M(r) 22,000 proteins being almost totally blocked. Other gene subfamilies were affected but to a lesser extent.

In this paper we characterize the effect of varying the solution conditions and filter-binding protocols on the extent and selectivity of DNA retention on nitrocellulose filters by DNA-binding proteins. These effects are illustrated by the binding interaction of Escherichia coli RNA polymerase with λ and T7 phage DNA restriction fragments. We present procedures which will help enhance the selective retention of some DNA restriction fragments over others. These include increasing the pH and salt concentration, decreasing the enzyme-to-DNA ratio, and including an appropriate washing step. Selective binding is not dependent on the presence of Mg2+. Although we only show data for RNA polymerase-DNA interactions, many of the principles discussed are likely to find practical applications in studying selective DNA-protein binding in general.

The nucleotide sequence of gz19ab11, a clone that corresponds to the coding and flanking sequences of an Mr 19,000 alpha zein, was determined. Comparison of the DNA sequences flanking this gene with those of other members of the gene subfamily showed that sequence conservation extends 820 nucleotides into the 5' flanking region and 130 nucleotides into the 3' flanking region. Southern blot analysis of maize DNA indicated that highly repetitive sequences are located within 950 bp 5' and 300 bp 3' to the protein coding region of these genes. The coding region of gz19ab11 is similar to but not identical with cDNA clones corresponding to Mr 19,000 zeins, and analysis of zein transcripts indicated that this gene is expressed exclusively in endosperm tissue. RNAs which correspond to transcripts originating 60 nucleotides, and more than 800 nucleotides, upstream of the initiation codon were detected for this and a related gene. However, the concentration of the large RNA species was several orders of magnitude less than that of the shorter RNAs. The functional significance of these large RNA transcripts in zein gene expression is unclear.