Mutational analysis of branching in pea. Evidence that Rms1 and Rms5 regulate the same novel signal.

The fifth increased branching ramosus (rms) mutant, rms5, from pea (Pisum sativum), is described here for phenotype and grafting responses with four other rms mutants. Xylem sap zeatin riboside concentration and shoot auxin levels in rms5 plants have also been compared with rms1 and wild type (WT). Rms1 and Rms5 appear to act closely at the biochemical or cellular level to control branching, because branching was inhibited in reciprocal epicotyl grafts between rms5 or rms1 and WT plants, but not inhibited in reciprocal grafts between rms5 and rms1 seedlings. The weakly transgressive or slightly additive phenotype of the rms1 rms5 double mutant provides further evidence for this interaction. Like rms1, rms5 rootstocks have reduced xylem sap cytokinin concentrations, and rms5 shoots do not appear deficient in indole-3-acetic acid or 4-chloroindole-3-acetic acid. Rms1 and Rms5 are similar in their interaction with other Rms genes. Reciprocal grafting studies with rms1, rms2, and rms5, together with the fact that root xylem sap cytokinin concentrations are reduced in rms1 and rms5 and elevated in rms2 plants, indicates that Rms1 and Rms5 may control a different pathway than that controlled by Rms2. Our studies indicate that Rms1 and Rms5 may regulate a novel graft-transmissible signal involved in the control of branching.

Pea Mutants with Reduced Sensitivity to Far-Red Light Define an Important Role for Phytochrome A in Day-Length Detection.

In garden pea (Pisum sativum L.), a long-day plant, long photoperiods promote flowering by reducing the synthesis or transport of a graft-transmissible inhibitor of flowering. Previous physiological studies have indicated that this promotive effect is predominantly achieved through a response that requires long exposures to light and for which far-red (FR) light is the most effective. These characteristics implicate the action of phytochrome A (phyA). To investigate this matter further, we screened ethylmethane sulfonate-mutagenized pea seedlings for FR-unresponsive, potentially phyA-deficient mutants. Two allelic, recessive mutants were isolated and were designated fun1 for FR unresponsive. The fun1-1 mutant is specifically deficient in the PHYA apoprotein and has a seedling phenotype indistinguishable from wild type when grown under white light. However, fun1-1 plants grown to maturity under long photoperiods show a highly pleiotropic phenotype, with short internodes, thickened stems, delayed flowering and senescence, longer peduncles, and higher seed yield. This phenotype results in large part from an inability of fun1-1 to detect day extensions. These results establish a crucial role for phyA in the control of flowering in pea, and show that phyA mediates responses to both red and FR light. Furthermore, grafting and epistasis studies with fun1 and dne, a mutant deficient in the floral inhibitor, show that the roles of phyA in seedling deetiolation and in day-length detection are genetically separable and that the phyA-mediated promotion of flowering results from a reduction in the synthesis or transport of the floral inhibitor.

Branching in Pea (Action of Genes Rms3 and Rms4).

The nonallelic ramosus mutations rms3-2 and rms4 of pea (Pisum sativum L.) cause extensive release of vegetative axillary buds and lateral growth in comparison with wild-type (cv Torsdag) plants, in which axillary buds are not normally released under the conditions utilized. Grafting studies showed that the expression of the rms4 mutation in the shoot is independent of the genotype of the root-stock. In contrast, the length of the branches at certain nodes of rms3-2 plants was reduced by grafting to wild-type stocks, indicating that the wild-type Rms3 gene may control the level of a mobile substance produced in the root. This substance also appears to be produced in the shoot because Rms3 shoots did not branch when grafted to mutant rms3-2 rootstocks. However, the end product of the Rms3 gene appears to differ from that of the Rms2 gene (C.A. Beveridge, J.J. Ross, and I.C. Murfet [1994] Plant Physiol 104: 953-959) because reciprocal grafts between rms3-2 and rms2 seedlings produced mature shoots with apical dominance similar to that of rms3-2 and rms2 shoots grafted to wild-type stocks. Indole-3-acetic acid levels were not reduced in apical or nodal portions of rms4 plants and were actually elevated (up to 2-fold) in rms3-2 plants. It is suggested that further studies with these branching mutants may enable significant progress in understanding the normal control of apical dominance and the related communication between the root and shoot.

New lv Mutants of Pea Are Deficient in Phytochrome B.

The lv-1 mutant of pea (Pisum sativum L.) is deficient in responses regulated by phytochrome B (phyB) in other species but has normal levels of spectrally active phyB. We have characterized three further lv mutants (lv-2, lv-3, and lv-4), which are all elongated under red (R) and white light but are indistinguishable from wild type under far-red light. The phyB apoprotein present in the lv-1 mutant was undetectable in all three new lv mutants. The identification of allelic mutants with and without phyB apoprotein suggests that Lv may be a structural gene for a B-type phytochrome. Furthermore, it indicates that the lv-1 mutation results specifically in the loss of normal biological activity of this phytochrome. Red-light-pulse and fluence-rate-response experiments suggest that lv plants are deficient in the low-fluence response (LFR) but retain a normal very-low-fluence-rate-dependent response for leaflet expansion and inhibition of stem elongation. Comparison of lv alleles of differing severity indicates that the LFR for stem elongation can be mediated by a lower level of phyB than the LFR for leaflet expansion. The retention of a strong response to continuous low-fluence-rate R in all four lv mutants suggests that there may be an additional phytochrome controlling responses to R in pea. The kinetics of phytochrome destruction and reaccumulation in the lv mutant indicate that phyB may be involved in the light regulation of phyA levels.

Branching Mutant rms-2 in Pisum sativum (Grafting Studies and Endogenous Indole-3-Acetic Acid Levels).

Isogenic lines of pea (Pisum sativum L.) were used to determine the physiological site of action of the Rms-2 gene, which maintains apical dominance, and its effect on endogenous free indole-3-acetic acid (IAA) levels. In mutant rms-2 scions, which normally produce lateral branches below node 3 and above node 7, apical dominance was almost fully restored by grafting to Rms-2 (wild-type) stocks. In the reciprocal grafts, rms-2 stocks did not promote branching in wild-type shoots. Together, these results suggest that the Rms-2 gene inhibits branching in the shoot of pea by controlling the synthesis of a translocatable (hormone-like) substance that is produced in the roots and/or cotyledons and in the shoot. At all stages, including the stage at which aerial lateral buds commence outgrowth, the level of IAA in rms-2 shoots was elevated (up to 5-fold) in comparison with that in wild-type shoots. The internode length of rms-2 plants was 40% less than in wild-type plants, and the mutant plants allocated significantly more dry weight to the shoot than to the root in comparison with wild-type plants. Grafting to wild-type stocks did not normalize IAA levels or internode length in rms-2 scions, even though it inhibited branching, suggesting that the involvement of Rms-2 in the control of IAA level and internode length may be confined to processes in the shoot.

Distribution of Gibberellins in Lathyrus odoratus L. and Their Role in Leaf Growth.

In sweet pea (Lathyrus odoratus L.) the mutant allele l reduced the level of gibberellin A1 (GA1) in expanding leaflets and resulted in smaller, more oval leaflets compared with the wild type. The apical portions of 6-d-old wild-type (L) seedlings also contained less GA1 and produced smaller, more oval leaflets than did comparable 20-d-old L seedlings. Application of GA1 markedly altered leaflet shape and, at certain dosages, restored the wild-type shape and size to leaflets of the l (dwarf) mutant. Taken together, these observations indicate that GA1 performs a regulatory role in the control of leaf growth in this species. The levels of GA1 precursors in the wild type were also determined. Rapidly expanding internodes contained much more gibberellin A19 (GA19) than gibberellin A20 (GA20), whereas the opposite was true for expanding leaflets. Although in entire apical portions of established seedlings the level of GA20 exceeded that of GA19, apical portions of very young seedlings contained more GA19 than GA20. Basal stem tissue of established seedlings also contained substantially more GA19 than GA20 or GA1. Both stems and leaflets from the basal portion of the plant contained much less GA20 and GA1 than did the rapidly expanding apical tissue. The implications of these results for the regulation of GA1 biosynthesis are discussed.

Flowering and branching in Lathyrus odoratus L.: loci sp and b.

A second flowering gene, Sp, which influences sensitivity to photoperiod, is identified in the sweet pea, Lathyrus odoratus L. Genes Sp and Dn (h) act in a complementary manner to confer the summer-flowering phenotype and a near obligate long day requirement for flowering in the unvernalized state. Mutations sp and Dn (i) each diminish the response to photoperiod, and genotypes sp Dn (h) and Sp Dn (i) confer a spring-flowering phenotype. Response to photoperiod is further reduced in genotype sp Dn (i), which flowers only marginally later than the day-neutral or winter-flowering phenotype characterized by genotypes Sp dn and sp dn (gene dn is epistatic to the gene pair Sp/sp). Like Dn (i), gene sp reduces basal branching, while a branching gene, here resymbolized b, is shown to delay flowering in certain circumstances. Gene dn largely prevents basal branching in either b or B plants, but dn b plants do produce lateral shoots from the upper nodes, leading to a novel phenotype. The implications of the interactions between genes sp, Dn (i), dn and b are discussed with respect to the control of flowering and branching.

Internode length in Pisum : The Le gene controls the 3ß-hydroxylation of gibberellin A20 to gibberellin A 1.

The influence of the Na and Le genes in peas on gibberellin (GA) levels and metabolism were examined by gas chromatographic-mass spectrometric analysis of extracts from a range of stem-length genotypes fed with [(13)C, (3)H]GA20. The substrate was metabolised to [(13)C, (3)H]GA1, [(13)C, (3)H]GA8 and [(13)C, (3)H]GA29 in the immature, expanding apical tissue of all genotypes carrying Le. In contrast, [(13)C, (3)H]GA29 and, in one line, [(13)C, (3)H]GA29-catabolite, were the only products detected in plants homozygous for the le gene. These results confirm that the Le gene in peas controls the 3ß-hydroxylation of GA20 to GA1. Qualitatively the same results were obtained irrespective of the genotype at the Na locus. In all Na lines the [(13)C, (3)H]GA20 metabolites were considerably diluted by endogenous [(12)C]GAs, implying that the metabolism of [(13)C, (3)H]GA20 mirrored that of endogenous [(12)C]GA20. In contrast, the [(13)C, (3)H]GA20 metabolites in na lines showed no dilution with [(12)C]GAs, confirming that the na mutation prevents the production of C19-GAs. Estimates of the levels of endogenous GAs in the apical tissues of Na lines, made from the (12)C:(13)C isotope ratios and the radioactivity recovered in respective metabolites, varied between 7 and 40 ng of each GA per plant in the tissue expanded during the 5 d between treatment with [(13)C, (3)H]GA20 and extraction. No [(12)C]GA1 and only traces of [(12)C]GA8 (in one line) were detected in the two Na le lines examined. These results are discussed in relation to recent observations on dwarfism in rice and maize.