Bruchins: insect-derived plant regulators that stimulate neoplasm formation.

Pea weevil (Bruchus pisorum L.) oviposition on pods of specific genetic lines of pea (Pisum sativum L.) stimulates cell division at the sites of egg attachment. As a result, tumor-like growths of undifferentiated cells (neoplasms) develop beneath the egg. These neoplasms impede larval entry into the pod. This unique form of induced resistance is conditioned by the Np allele and mediated by a recently discovered class of natural products that we have identified from both cowpea weevil (Callosobruchus maculatus F.) and pea weevil. These compounds, which we refer to as "bruchins," are long-chain alpha,omega-diols, esterified at one or both oxygens with 3-hydroxypropanoic acid. Bruchins are potent plant regulators, with application of as little as 1 fmol (0.5 pg) causing neoplastic growth on pods of all of the pea lines tested. The bruchins are, to our knowledge, the first natural products discovered with the ability to induce neoplasm formation when applied to intact plants.

Gibberellins and seed development in maize. I. Evidence that gibberellin/abscisic acid balance governs germination versus maturation pathways.

Abscisic acid (ABA) is required for the regulation of seed maturation in maize (Zea mays L.). Mutants blocked in ABA synthesis (such as viviparous-5) do not mature to quiescent, desiccation-tolerant seeds, but germinate on the ear midway through kernel development. Because gibberellins (GA) and ABA act antagonistically in many aspects of plant development, we hypothesized that ABA antagonizes a positive GA signal for precocious germination in maize. In these experiments, we show that a GA deficiency early in seed development, induced genetically or via biosynthesis inhibitors, suppresses vivipary in ABA-deficient developing kernels. The resulting seeds have both desiccation tolerance and storage longevity. Temporal analysis of GA accumulation in wild-type kernels revealed the accumulation of bioactive GA(1) and GA(3) prior to the peak in ABA content. We speculate that these GAs stimulate a developmental program leading to vivipary in the absence of normal amounts of ABA, and that a reduction of GA content re-establishes an ABA/GA ratio appropriate for suppression of germination and induction of maturation. In contrast, the induction of a GA deficiency did not suppress vivipary in viviparous-1 mutant kernels, suggesting that VP1 acts downstream of both GA and ABA in programming seed development.

The SLENDER gene of pea encodes a gibberellin 2-oxidase.

The amount of active gibberellin (GA) in plant tissues is determined in part by its rate of catabolism through oxidation at C-2. In pea (Pisum sativum L.) seeds, GA 2-oxidation is controlled by the SLN (SLENDER) gene, a mutation of which produces seedlings characterized by a slender or hyper-elongated phenotype. We cloned a GA 2-oxidase cDNA from immature pea seeds by screening an expression library for enzyme activity. The clone contained a full-length open reading frame encoding a protein of 327 amino acids. Lysate of bacterial cultures expressing the protein converted the C(19)-GAs, GA(1), GA(4), GA(9), and GA(20) to the corresponding 2beta-hydroxy products. GA(9) and GA(20) were also converted to GA(51) and GA(29) catabolites, respectively. The gene appeared to be one member of a small family of GA 2-oxidases in pea. Transcript was found predominantly in roots, flowers, young fruits, and testae of seeds. The corresponding transcript from sln pea contained a point mutation and did not produce active enzyme when expressed heterologously. RFLP analysis of a seedling population segregating for SLN and sln alleles showed the homozygous mutant allele co-segregating with the characteristic slender phenotype. We conclude that SLN encodes GA 2-oxidase.

Mendel's dwarfing gene: cDNAs from the Le alleles and function of the expressed proteins.

The major gibberellin (GA) controlling stem elongation in pea (Pisum sativum L.) is GA1, which is formed from GA20 by 3beta-hydroxylation. This step, which limits GA1 biosynthesis in pea, is controlled by the Le locus, one of the original Mendelian loci. Mutations in this locus result in dwarfism. We have isolated cDNAs encoding a GA 3beta-hydroxylase from lines of pea carrying the Le, le, le-3, and led alleles. The cDNA sequences from le and le-3 each contain a base substitution resulting in single amino acid changes relative to the sequence from Le. The cDNA sequence from led, a mutant derived from an le line, contains both the le "mutation" and a single-base deletion, which causes a shift in reading frame and presumably a null mutation. cDNAs from each line were expressed in Escherichia coli. The expression product for the clone from Le converted GA9 to GA4, and GA20 to GA1, with Km values of 1.5 microM and 13 microM, respectively. The amino acid substitution in the clone from le increased Km for GA9 100-fold and reduced conversion of GA20 to almost nil. Expression products from le and le-3 possessed similar levels of 3beta-hydroxylase activity, and the expression product from led was inactive. Our results suggest that the 3beta-hydroxylase cDNA is encoded by Le. Le transcript is expressed in roots, shoots, and cotyledons of germinating pea seedlings, in internodes and leaves of established seedlings, and in developing seeds.

Feed-back regulation of gibberellin biosynthesis and gene expression in Pisum sativum L.

Treatment of tall and dwarf (3 beta-hydroxylase impaired) genotypes of pea (Pisum sativum L.) with the synthetic, highly active gibberellin (GA), 2,2-dimethyl GA4, reduced the shoot contents of C19-GAs, including GA1, and increased the concentration of the C20-GA, GA19. In shoots of the slender (la crys) mutant, the content of C19-GAs was lower and GA19 content was higher than in those of the tall line. Metabolism of GA19 and GA20 in leaves of a severe (na) GA-deficient dwarf mutant was reduced by GA treatment. The results suggest feed-back regulation of the 20-oxidation and 3 beta-hydroxylation reactions. Feed-back regulation of GA 20-oxidation was studied further using a cloned GA 20-oxidase cDNA from pea. The cDNA, Ps074, was isolated using polymerase chain reaction with degenerate oligonucleotide primers based on pumpkin and Arabidopsis 20-oxidase sequences. After expression of this cDNA clone in Escherichia coli, the product oxidized GA12 to GA15, GA24 and the C19-GA, GA9, which was the major product. The 13-hydroxylated substrate GA53 was similarly oxidized, but less effectively than GA12, giving mainly GA44 with low yields of GA19 and GA20. Ps074 hybridized to polyadenylated RNA from expanding shoots of pea. Amounts of this transcript were less in the slender genotype than in the tall line and were reduced in GA-deficient genotypes by treatment with GA3, suggesting that there is feed-back regulation of GA 20-oxidase gene expression.

Response ofNp mutant of pea (Pisum sativum L.) to pea weevil (Bruchus pisorum L.) oviposition and extracts.

TheNp mutant of pea (Pisum sativum L.) is characterized by two physiological responses: growth of callus under pea weevil (Bruchus pisorum L., Coleoptera: Bruchidae) oviposition on pods, and formation of neoplastic callus on pods of indoor-grown plants. Although these two responses are conditioned byNp, they are anatomically and physiologically distinguishable, based on sites of origin, distribution pattern, and sensitivity to plant hormones. Further characterization of the response to extracts of pea weevil showed that response of excised pods, measured by callus formation, was log-linear, and treatment with as little as 10(-4) weevil equivalents produced a detectable response. Mated and unmated females contained similar amounts of callus-inducing compound(s), and immature females contained significantly less of the compound(s). Female vetch bruchids (Bruchus brachialis F., Coleoptera: Bruchidae), a related species, contained callus-inducing compound(s), but usually less than pea weevils on a per weevil basis. Males of both species contained less than 10% of the activity of the mature females. Extracts of female black vine weevils, a nonbruchid species, did not stimulate callus formation. Based on partitioning and TLC analysis, the biologically active constitutent(s) was stable and nonpolar. Thus, theNp allele probably conditions sensitivity to a nonpolar component of pea weevil oviposition as a mechanism of resistance to the weevil.

RNA-mediated virus resistance in transgenic plants: exploitation of a cellular pathway possibly involved in RNA degradation.

Transgenic Nicotiana tabacum cv. Burley 49 plants were generated that express the 5' untranslated region of the tobacco etch potyvirus (TEV) genome ligated to a mutated version of the TEV coat protein gene sequence that rendered it untranslatable. Eight different transgenic plant lines were analyzed for transgene expression and for resistance to TEV. Three different responses were noted when the transgenic plant lines were inoculated with TEV: 1) some were highly resistant, and no virus replication occurred; 2) some were susceptible but able to recover from systemic TEV infection; and 3) some were susceptible to TEV infection. Plant tissue displaying the recovery phenotype was analyzed for virus replication and transgene expression. Recovered tissue could not be infected with TEV and had steady-state transgene RNA levels which were five- to eightfold lower than those of unchallenged transgenic plant tissue. Nuclear runoff assays suggested a post-transcriptional reduction in specific RNA levels. The highly resistant and recovery phenotypes associated with TEV challenge inoculation and the reduction of steady-state RNA levels in recovered transgenic leaf tissue may be manifestations of a common mechanism.

Induction of a Highly Specific Antiviral State in Transgenic Plants: Implications for Regulation of Gene Expression and Virus Resistance.

Transgenic tobacco plants expressing either a full-length form of the tobacco etch virus (TEV) coat protein or a form truncated at the N terminus of the TEV coat protein were initially susceptible to TEV infection, and typical systemic symptoms developed. However, 3 to 5 weeks after a TEV infection was established, transgenic plants "recovered" from the TEV infection, and new stem and leaf tissue emerged symptom and virus free. A TEV-resistant state was induced in the recovered tissue. The resistance was virus specific. Recovered plant tissue could not be infected with TEV, but was susceptible to the closely related virus, potato virus Y. The resistance phenotype was functional at the single-cell level because protoplasts from recovered transgenic tissue did not support TEV replication. Surprisingly, steady state transgene mRNA levels in recovered tissue were 12-to 22-fold less than transgene mRNA levels in uninoculated transgenic tissue of the same developmental stage. However, nuclear run-off studies suggested that transgene transcription rates in recovered and uninoculated plants were similar. We propose that the resistant state and reduced steady state levels of transgene transcript accumulation are mediated at the cellular level by a cytoplasmic activity that targets specific RNA sequences for inactivation.

Gibberellin concentration and transport in genetic lines of pea : effects of grafting.

Effects of the Na and Le loci on gibberellin (GA) content and transport in pea (Pisum sativum L.) shoots were studied. GA(1), GA(8), GA(17), GA(19), GA(20), GA(29), GA(44), GA(8) catabolite, and GA(29) catabolite were identified by full-scan gas chromatography-mass spectrometry in extracts of expanding and fully expanded tissues of line C79-338 (Na Le). Quantification of GAs by gas chromatography-single-ion monitoring using deuterated internal standards in lines differing at the Na and Le alleles showed that na reduced the contents of GA(19), GA(20), and GA(29) on average to <3% and of GA(1) and GA(8) to <30% of those in corresponding Na lines. In expanding tissues from Na le lines, GA(1) and GA(8) concentrations were reduced to approximately 10 and 2%, respectively, and GA(29) content increased 2- to 3-fold compared with those in Na Le plants. There was a close correlation between stem length and the concentrations of GA(1) or GA(8) in shoot apices in all six genotypes investigated. In na/Na grafts, internode length and GA(1) concentration of nana scions were normalized, the GA(20) content increased slightly, but GA(19) levels were unaffected. Movement of labeled GAs applied to leaves on Na rootstocks indicated that GA(19) was transported poorly to apices of na scions compared with GA(20) and GA(1). Our evidence suggests that GA(20) is the major transported GA in peas.

Development of the alt Mutant of Pisum sativum L.

The alt (albina-terminalis) mutant of Pisum sativum L. germinates normally, produces several nodes, and then above a sharp transition produces 2 to 3 bleached nodes, ceases growth, and eventually dies. Green nodes have normal chlorophyll content, absorption spectra, photosynthetic rates, and ultrastructure. In bleaching tissues, the chloroplasts degenerate rapidly, followed by extensive disruption and loss of the remaining cytoplasm and organelles. Application of tissue extracts of normal genotypes of pea, corn, and bean stimulates apical development of alt. The resulting tissues have essentially normal structure and function. Application of thiamine, thiamine monophosphate, and thiamine pyrophosphate also stimulate normal apical development at concentrations of 1 micromolar and above. Partial characterization of the stimulus from pea seed extracts is consistent with thiamine as the active factor.

Genetic Regulation of Flowering in Grafts on Pisum sativum L.

Lf, E, Sn, and Hr are major loci that condition the flowering and photoperiod responses of Pisum sativum L. Genetic lines containing the dominant alleles of these loci are characterized by flowering in long days, but having a prolonged (>50 node) vegetative phase in short days. A representative of this class, response type G, was used as a receptor in short days for donors of other flowering response types. The qualitative and quantitative flowering response of G receptors depended on the genotype of the donor. Donors containing sn hr induced the earliest development, followed by sn Hr and Sn hr donors. The Lf and E loci in foliar donors apparently did not affect flowering of G. Five-leaved > single-leaved > cotyledonary donors in effecting a flowering response in G, in part due to the longer life of the foliar donors. The responses of G to the various donors were generally consistent with the proposed roles of Lf, E, and Sn, but the role of Hr in these grafts was unclear.

The endogenous gibberellins of vegetative and reproductive tissue of G2 peas.

The gibberellins (GAs) of both vegetative (leaves and stems) and reproductive (pods and seeds) tissue of the G2 strain of peas Pisum sativum L. were characterized in purified extracts by a combination of sequential silicic-acid partition column chromatography, and gas chromatography-mass spectrometry. Gibberellins A19, A20, A29 and an A29 catabolite were identified in both types of tissue. Gibberellins A9, A17 and A44 were also found in pods and seeds.

Photoperiod-induced changes in gibberellin metabolism in relation to apical growth and senescence in genetic lines of peas (Pisum sativum L.).

In an early-flowering line of pea (G2) apical senescence occurs only in long days (LD), while growth in short days (SD) is indeterminate. In SD, G2 plants are known to produce a graft-transmissible substance which delays apical senescence in related lines that are photoperiod-insensitive with regard to apical senescence. Gibberellic acid (GA3) applied to the apical bud of G2 plants in LD delayed apical senescence indefinitely, while N(6)-benzyladenine and α-naphthaleneacetic acid were ineffective. Of the gibberellins native to pea, GA9 had no effect whereas GA20 had a moderate senescence-delaying effect. [(3)H]GA9 metabolism in intact leaves of G2 plants was inhibited by LD and was restored by placing the plants back in SD. Leaves of photoperiod-insensitive lines (I-types) metabolized GA9 readily regardless of photoperiod, but the metabolites differed qualitatively from those in G2 leaves. A polar GA9 metabolite, GAE, was found only in G2 plants in SD. The level of GA-like substances in methanol extracts from G2 plants dropped about 10-fold after the plants were moved from SD to LD; it was restored by transferring the plants back to SD. A polar zone of these GA-like materials co-chromatographed with GAE. It is suggested that a polar gibberellin is synthesized by G2 plants in SD; this gibberellin promotes shoot growth and meristematic activity in the shoot apex, preventing senescence.

Evidence for a graft-transmissible substance which delays apical senescence in Pisum sativum L.

Apical senescence in an early flowering line of pea, G2, is greatly delayed by short days. This behavior is controlled by two dominant genes. Apical senescence of ungrafted, insensitive (I) lines is unaffected by photoperiod. When I-type scions with one of the two required genes were grafted onto G2, apical senescence of the I-type was delayed in short days, but not in long days. Flowering of the I-type was unaffected. The apex of the G2 stock was unaffected as well. Apical senescence of an I-type line lacking both photoperiod genes was not delayed when grafted on G2 in short days. It is concluded that G2 plants grown in short days produce a graft-transmissible factor which delays apical senescence of photoperiodically insensitive lines.

Photoperiodic control of apical senescence in a genetic line of peas.

An early flowering genetic line of peas (Pisum sativum L.), designated G2, has dominant genes at two different loci, both of which function in short days to greatly extend the reproductive phase and thus to delay apical senescence. Long days (18 hours) promote senescence in this line, but the effect is reversible by reinstatement of short days (9 hours) until 3 to 4 days before the apex senesces. The response to photoperiod was quantitative. Increasing the photoperiod from 14 to 18 hours led to a progressive decrease in the number of nodes formed prior to death of the apex. Induction of senescence was determined by the total number of hours of light and darkness rather than by the length of the dark period. Senescence required flower and fruit development as well as long days.