Carl D. Schlagnhaufer

A fruiting body tissue method for efficient Agrobacterium-mediated transformation of Agaricus bisporus.

ABSTRACT We describe a modified Agrobacterium-mediated method for the efficient transformation of Agaricus bisporus. Salient features of this procedure include cocultivation ofAgrobacterium and fruiting body gill tissue and use of a vector with a homologous promoter. This method offers new prospects for the genetic manipulation of this commercially important mushroom species.

Identification and characterization of a full-length cDNA encoding for an auxin-induced 1-aminocyclopropane-1-carboxylate synthase from etiolated mung bean hypocotyl segments and expression of its mRNA in response to indole-3-acetic acid

Abstract1-Aminocyclopropane-1-carboxylate (ACC) synthase (EC 4.4.1.14) is the key regulatory enzyme in the ethylene biosynthetic pathway. The identification and characterization of a full-length cDNA (pAIM-1) 1941 bp in length for indole-3-acetic acid (IAA)-induced ACC synthase is described in this paper. The pAIM-1 clone has an 87 bp leader and a 402 bp trailing sequence. The open reading frame is 1452 bp long encoding for a 54.6 kDa polypeptide (484 amino acids) which has a calculated isoelectric point of 6.0. In vitro transcription and translation experiments support the calculated molecular weight and show that the enzyme does not undergo processing. Eleven of the twelve amino acid residues which are conserved in aminotransferases are found in pAIM-1. The sequence for pMAC-1 which is one of the 5 genes we have identified in mung bean is contained in pAIM-1. pAIM-1 shares between 52 to 65% homology with previously reported sequences for ACC synthase at the protein level. There is little detectable pAIM-1 message found in untreated mung bean tissues; however, expression is apparent within 30 min following the addition of 10 μM IAA reaching a peak after approximately 5 h with a slight decrease in message after 12 h. These changes in message correlate with changes in ACC levels found in these tissues following treatment with 10 μM IAA.

Ozone-Induced Ethylene Emission Accelerates the Loss of Ribulose-1,5-Bisphosphate Carboxylase/Oxygenase and Nuclear-Encoded mRNAs in Senescing Potato Leaves

The relationships among O3-induced accelerated senescence, induction of ethylene, and changes in specific mRNA and protein levels were investigated in potato (Solanum tuberosum L. cv Norland) plants. When plants were exposed to 0.08 [mu]L L-1 O3 for 5 h d-1, steady-state levels of rbcS mRNA declined at least 5-fold in expanding leaves after 3 d of O3 exposure and ethylene levels increased 6- to 10-fold. The expression of OIP-1, a 1-aminocyclopropane-1-carboxylate synthase cDNA from potato, correlated with increased production of ethylene and decreased levels of rbcS mRNA in foliage of plants treated with O3. In plants exposed to 0.30 [mu]L L-1 O3 for 4 h, rbcS transcript levels were reduced 4-fold, whereas nuclear run-on experiments revealed that rbcS transcription declined an average of 50%. The loss of rbcS mRNA may be due, in part, to posttranscriptional regulation. The levels of transcripts for other chloroplast proteins, glyceraldehyde-3-phosphate dehydrogenase, and a photosystem II chlorophyll a/b-binding protein decreased in O3-treated plants, in parallel with the decrease in rbcS mRNA. The steady-state mRNA level of a cytosolic glyceral-dehyde-3-phosphate dehydrogenase increased in O3-treated plants. The induction of ethylene and changes in transcript levels preceded visible leaf damage and decreases in ribulose-1,5-bisphosphate carboxylase/oxygenase protein levels.

Sequential expression of two 1-aminocyclopropane-1-carboxylate synthase genes in response to biotic and abiotic stresses in potato (Solanum tuberosum L.) leaves.

Plants produce ethylene in response to many biotic and abiotic stresses. In response to ozone the foliage of potato plants sequentially expressed two ACC synthase genes (ST-ACS4, ST-ACS5). The same expression pattern of the two genes also occurred in response to Cu2+ and infection with Alternaria solani. ST-ACS5 expression increases very rapidly reaching a maximum earlier than ST-ACS4 transcripts, after which ST-ACS5 expression declines. ST-ACS4 expression increases at a slower rate and reaches its maximum after ST-ACS5. The sequential nature of expression argues that the two genes have different signal transduction and gene regulatory mechanisms.

Identification of two new members of the l-aminocyclopropane-l-carboxylate synthase-encoding multigene family in mung bean

The key enzyme regulating ethylene biosynthesis in higher plants is 1-aminocyclopropane-1-carboxylate (ACC) synthase. In mung bean (MB), the existence of three genes encoding this enzyme has previously been reported [Botella et al., Plant Mol. Biol. 18 (1992) 793-797], one of which corresponds to a full-length indole-3-acetic acid-inducible cDNA [Botella et al., Plant Mol. Biol. (1992) 425-436]. In this paper we report the cloning of two new genomic sequences coding for ACC synthase in MB (MAC-4 and MAC-5). MAC-4 is 1340 bp long and encodes 388 amino acids (aa) while MAC-5 is 1393 bp long and encodes for 391 aa. Genomic Southern analysis suggests the existence of only one copy of each gene in the genome.

Molecular cloning of an ozone-induced 1-aminocyclopropane-1-carboxylate synthase cDNA and its relationship with a loss of rbcS in potato (Solanum tuberosum L.) plants

Acute or chronic exposure of potato plants to ozone (O3) induces ethylene production. We isolated a 1586 bp cDNA (pOIP-1) encoding 1-aminocyclopropane-1-carboxylate (ACC) synthase from a cDNA library constructed with mRNA extracted from O3-treated leaves. The clone has a 1365 bp open reading frame and a 221 bp trailing sequence. The active site found in all ACC synthases and 11 of the 12 amino acid residues conserved in aminotransferases are found in pOIP-1. Northern analysis showed that the mRNA encoding ACC synthase was detectable 1 h after the onset of O3 exposure, and the message increased over time as did ethylene production. Concurrent with the increased ACC synthase mRNA was a decrease in the message for the Rubisco small subunit (rbcS) with no change in the large subunit (rbcL). When the plants were treated with aminooxyacetic acid (AOA), both ethylene production and level of ACC synthase transcript were inhibited. The decline in rbcS was also inhibited by AOA suggesting a correlation between ethylene production and loss of rbcS. Based on nuclear run-on studies it appears that the increase in ACC synthase mRNA may result from O3-induced transcriptional activity.

The uptake and metabolism of brassinosteroid by tomato (Lycopersicon esculentum) plants

Summary When brassinosteroids (BR) are applied to the roots of hydroponically grown tomato plants there is an accumulation of 1-aminocyclopropane-l-carboxylic acid (ACQ in the leaves. In this work, BR-induced ethylene production in tomato plants was studied further using 3 HBR. Twenty day old tomato plants were incubated in 25 mL Hoagland’s solution plus 1.1 µMBR and 3.7 x 10 6 cpm of 3 HBR. During incubation the tomato plants accumulated ACC and extractable radioactivity. The 24-h plant extracts contained 2695 nmolkg -1 (fresh mass basis) of ACC compared with 73 nmolkg -1 in the control. The 24-h treated plant extracts also contained over 2.12 × 10 6 cpm kg -1 of tritium. Migration of the radioactivity on silica gel TLC plates showed there was no apparent metabolism of BR during the first four hours; however, by 8 h there were two radioactive spots on the plate. The 12-h extract contained 3 radioactive spots, the original BR plus two metabolic products. When plants were incubated with BR for 24 h and transferred to BR-free Hoaglands solution, the ACC content of the tissues decreased and there was an increase in the amount of tritium migrating as BR metabolites. The plant apparently metabolizes BR to inactive forms, and the overproduction of ethylene ceases. The identities of these metabolic products are unknown at this time.

Identification and characterization of three putative genes for 1-aminocyclopropane-1-carboxylate synthase from etiolated mung bean hypocotyl segments.

The polymerase chain reaction (PCR) was used to produce 3 putative clones for ACC synthase from etiolated mung bean (Vigna radiata Rwilcz cv. Berken) hypocotyls. This was accomplished by utilizing genomic DNA from mung bean and degenerate primers made from information derived from highly conserved regions of ACC synthase from different plant tissues. The total length of pMAC-1, pMAC-2 and pMAC-3 are 308, 321, and 326 bp, respectively, all of which code for 68 amino acids. The introns for pMAC-1, pMAC-2 and pMAC-3 are 92, 105, and 110 bp, respectively. The degrees of homology at the DNA level for each of these clones is ca. 80% in their coding region and ca. 50% in their respective introns. This is the first report providing evidence that there are at least 3 genes for ACC synthase in etiolated mung bean.

Long-Distance Translocation of Protein during Morphogenesis of the Fruiting Body in the Filamentous Fungus, Agaricus bisporus

Commercial cultivation of the mushroom fungus, Agaricus bisporus, utilizes a substrate consisting of a lower layer of compost and upper layer of peat. Typically, the two layers are seeded with individual mycelial inoculants representing a single genotype of A. bisporus. Studies aimed at examining the potential of this fungal species as a heterologous protein expression system have revealed unexpected contributions of the mycelial inoculants in the morphogenesis of the fruiting body. These contributions were elucidated using a dual-inoculant method whereby the two layers were differientially inoculated with transgenic β-glucuronidase (GUS) and wild-type (WT) lines. Surprisingly, use of a transgenic GUS line in the lower substrate and a WT line in the upper substrate yielded fruiting bodies expressing GUS activity while lacking the GUS transgene. Results of PCR and RT-PCR analyses for the GUS transgene and RNA transcript, respectively, suggested translocation of the GUS protein from the transgenic mycelium colonizing the lower layer into the fruiting body that developed exclusively from WT mycelium colonizing the upper layer. Effective translocation of the GUS protein depended on the use of a transgenic line in the lower layer in which the GUS gene was controlled by a vegetative mycelium-active promoter (laccase 2 and β-actin), rather than a fruiting body-active promoter (hydrophobin A). GUS-expressing fruiting bodies lacking the GUS gene had a bonafide WT genotype, confirmed by the absence of stably inherited GUS and hygromycin phosphotransferase selectable marker activities in their derived basidiospores and mycelial tissue cultures. Differientially inoculating the two substrate layers with individual lines carrying the GUS gene controlled by different tissue-preferred promoters resulted in up to a ∼3.5-fold increase in GUS activity over that obtained with a single inoculant. Our findings support the existence of a previously undescribed phenomenon of long-distance protein translocation in A. bisporus that has potential application in recombinant protein expression and biotechnological approaches for crop improvement.

Mushroom (Agaricus bisporus).

We have devised an easy and effective genetic transformation method for the preeminent edible mushroom, Agaricus bisporus. Our method exploits the T-DNA transfer mechanism in Agrobacterium tumefaciens and relies on the reproductive fruiting body as the recipient tissue. The use of fruiting body explants, particularly the gill, provided high-frequency transformation, overcoming the inefficacy of Agrobacterium-based methods targeting fungal spores or vegetative mycelium. The protocol entails incubation of A. tumefaciens for 3 h with acetosyringone, a signaling molecule that launches the gene transfer mechanism, co-cultivation of the induced bacterium and gill explants for 3 d, and selection for transformants based on an inherited resistance to the antibiotic hygromycin. Between 7 and 28 d on the selection medium, upwards of 95% of the gill explants generate hygromycin-resistant colonies. About 75% of the mushroom transformants show a single-copy of the hygromycin-resistant gene integrated at random sites in the genome.