Page W. Morgan

Programmed cell death and aerenchyma formation in roots

Lysigenous aerenchyma contributes to the ability of plants to tolerate low-oxygen soil environments, by providing an internal aeration system for the transfer of oxygen from the shoot. However, aerenchyma formation requires the death of cells in the root cortex. In maize, hypoxia stimulates ethylene production, which in turn activates a signal transduction pathway involving phosphoinositides and Ca2+. Death occurs in a predictable pattern, is regulated by a hormone (ethylene) and provides an example of programmed cell death.

Transduction of an Ethylene Signal Is Required for Cell Death and Lysis in the Root Cortex of Maize during Aerenchyma Formation Induced by Hypoxia

Ethylene has been implicated in signaling cell death in the lysigenous formation of gas spaces (aerenchyma) in the cortex of adventitious roots of maize (Zea mays) subjected to hypoxia. Various antagonists that are known to modify particular steps in signal transduction in other plant systems were applied at low concentrations to normoxic and hypoxic roots of maize, and the effect on cell death (aerenchyma formation) and the increase in cellulase activity that precedes the appearance of cell degeneration were measured. Both cellulase activity and cell death were inhibited in hypoxic roots in the presence of antagonists of inositol phospholipids, Ca2+- calmodulin, and protein kinases. By contrast, there was a parallel promotion of cellulase activity and cell death in hypoxic and normoxic roots by contact with reagents that activate G-proteins, increase cytosolic Ca2+, or inhibit protein phosphatases. Most of these reagents had no effect on ethylene biosynthesis and did not arrest root extension. These results indicate that the transduction of an ethylene signal leading to an increase in intracellular Ca2+ is necessary for cell death and the resulting aerenchyma development in roots of maize subjected to hypoxia.

The Sorghum Photoperiod Sensitivity Gene, Ma3, Encodes a Phytochrome B'

The Ma3 gene is one of six genes that regulate the photoperiodic sensitivity of flowering in sorghum (Sorghum bicolor [L.] Moench). The ma3R mutation of this gene causes a phenotype that is similar to plants that are known to lack phytochrome B, and ma3R sorghum lacks a 123-kD phytochrome that predominates in light-grown plants and that is present in non-ma3R plants. A population segregating for Ma3 and ma3R was created and used to identify two randomly amplified polymorphic DNA markers linked to Ma3. These two markers were cloned and mapped in a recombinant inbred population as restriction fragment length polymorphisms. cDNA clones of PHYA and PHYC were cloned and sequenced from a cDNA library prepared from green sorghum leaves. Using a genome-walking technique, a 7941-bp partial sequence of PHYB was determined from genomic DNA from ma3R sorghum. PHYA, PHYB, and PHYC all mapped to the same linkage group. The Ma3- linked markers mapped with PHYB more than 121 centimorgans from PHYA and PHYC. A frameshift mutation resulting in a premature stop codon was found in the PHYB sequence from ma3R sorghum. Therefore, we conclude that the Ma3 locus in sorghum is a PHYB gene that encodes a 123-kD phytochrome.

Ethylene Biosynthesis during Aerenchyma Formation in Roots of Maize Subjected to Mechanical Impedance and Hypoxia

Germinated maize (Zea mays L.) seedlings were enclosed in modified triaxial cells in an artificial substrate and exposed to oxygen deficiency stress (4% oxygen, hypoxia) or to mechanical resistance to elongation growth (mechanical impedance) achieved by external pressure on the artificial substrate, or to both hypoxia and impedance simultaneously. Compared with controls, seedlings that received either hypoxia or mechanical impedance exhibited increased rates of ethylene evolution, greater activities of 1-aminocyclopropane-1-carboxylic acid (ACC) synthase, ACC oxidase, and cellulase, and more cell death and aerenchyma formation in the root cortex. Effects of hypoxia plus mechanical impedance were strongly synergistic on ethylene evolution and ACC synthase activity; cellulase activity, ACC oxidase activity, or aerenchyma formation did not exhibit this synergism. In addition, the lag between the onset of stress and increases in both ACC synthase activity and ethylene production was shortened by 2 to 3 h when mechanical impedance or impedance plus hypoxia was applied compared with hypoxia alone. The synergistic effects of hypoxia and mechanical impedance and the earlier responses to mechanical impedance than to hypoxia suggest that different mechanisms are involved in the promotive effects of these stresses on maize root ethylene biosynthesis.

Phytochrome B Affects Responsiveness to Gibberellins in Arabidopsis

Plant responses to red and far-red light are mediated by a family of photoreceptors called phytochromes. Arabidopsis thaliana seedlings lacking one of the phytochromes, phyB, have elongated hypocotyls and other tissues, suggesting that they may have an alteration in hormone physiology. We have studied the possibility that phyB mutations affect seedling gibberellin (GA) perception and metabolism by testing the responsiveness of wild-type and phyB seedlings to exogenous GAs. The phyB mutant elongates more than the wild type in response to the same exogenous concentrations of GA3 or GA4, showing that the mutation causes an increase in responsiveness to GAs. Among GAs that we were able to detect, we found no significant difference in endogenous levels between wild-type and phyB mutant seedlings. However, GA4 levels were below our limit of detectability, and the concentration of that active GA could have varied between wild-type and phyB mutant seedlings. These results suggest that, although GAs are required for hypocotyl cell elongation, phyB does not act primarily by changing total seedling GA levels but rather by decreasing seedling responsiveness to GAs.

Induction of Enzymes Associated with Lysigenous Aerenchyma Formation in Roots of Zea mays during Hypoxia or Nitrogen Starvation

Either hypoxia, which stimulates ethylene biosynthesis, or temporary N starvation, which depresses ethylene production, leads to formation of aerenchyma in maize (Zea mays L.) adventitious roots by extensive lysis of cortical cells. We studied the activity of enzymes closely involved in either ethylene formation (1-amino-cyclopropane-1-carboxylic acid synthase [ACC synthase]) or cell-wall dissolution (cellulase). Activity of ACC synthase was stimulated in the apical zone of intact roots by hypoxia, but not by anoxia or N starvation. However, N starvation, as well as hypoxia, did enhance cellulase activity in the apical zone, but not in the older zones of the same roots. Cellulase activity did not increase during hypoxia or N starvation in the presence of aminoethoxyvinylglycine, an inhibitor of ACC synthase, but this inhibition of cellulase induction was reversed during simultaneous exposure to exogenous ethylene. Together these results indicate both the role of ethylene in signaling cell lysis in response to two distinct environmental factors and the significance of hypoxia rather than anoxia in stimulation of ethylene biosynthesis in maize roots.

Genetic Regulation of Development in Sorghum bicolor (VIII. Shoot Growth, Tillering, Flowering, Gibberellin Biosynthesis, and Phytochrome Levels Are Differentially Affected by Dosage of the ma3R Allele

Sorghum [Sorghum bicolor (L.) Moench] homozygous for ma3R lacks a type II, light-stable phytochrome of 123 kD and has a number of phenotypic characteristics consistent with the absence of functional phytochrome B. We have used plants heterozygous at Ma3 (Ma3/ma3R and ma3/ma3R) to determine the effect of dosage of ma3R on plant growth, flowering, gibberellin (GA) levels, and content of the 123-kD phytochrome. Both Ma3/ma3R and ma3/ma3R produced the same number of tillers per plant as their respective homozygous non-ma3R parents. Height of the heterozygotes was intermediate between the homozygous parents, although it was more similar to the non-ma3R genotypes. In both field and growth-chamber environments, the timing of floral initiation and anthesis in the heterozygotes also was intermediate, again more similar to non-ma3R plants. In Ma3/ma3R, levels of GA53, GA19, GA20, and GA1 were almost exactly intermediate between levels detected in Ma3/Ma3 and ma3R/ma3R plants. Immunoblot analysis indicated that there was less of the 123-kD phytochrome in Ma3/ma3R than in homozygous Ma3, whereas none was detected in ma3R/ma3R. The degree of dominance of Ma3 and ma3 over ma3R varies with phenotypic trait, indicating that mechanisms of activity of the 123-kD phytochrome vary among the biochemical processes involved in each phenotypic character. Although the heterozygotes were similar to homozygous Ma3 and ma3 plants in growth and flowering behavior, Ma3/ma3R contained 50% less of the bioactive GA (GA1) than non-ma3R genotypes. Thus, sensitivity to endogenous GAs also may be regulated by the 123-kD phytochrome. To fully regulate plant growth and development, two copies of Ma3 or ma3 are required to produce sufficient quantities of the light-stable, 123-kD phytochrome.

Is ethylene the Natural Regulator of Abscission

Ethylene and abscission have been linked in the literature for many decades, but only recently has ethylene’s role been supported by critical evidence. Thus it seems almost paradoxical to consider whether ethylene is the regulator of abscission. The question is made appropriate by attention to the involvement of ethylene in a natural abscission and to the mechanism(s) by which ethylene initiates abscission.

Effect of Gibberellin Biosynthesis Inhibitors on Native Gibberellin Content, Growth and Floral Initiation in Sorghum bicolor

Abstract. CCC, uniconazol, ancymidol, prohexadione-calcium (BX-112), and CGA 163′935, which represent three groups of gibberellin (GA) biosynthesis inhibitors, were applied as a soil drench to Sorghum bicolor cultivars 58M (phyB-1, phytochrome B-deficient mutant) and 90M (phyB-2, equivalent phenotypically to wild type, PHYB, except for small differences in flowering dates). The inhibitors that block steps before GA12 (CCC, uniconazol, and ancymidol) lowered the concentrations of all endogenous early-C13α-hydroxylation pathway GAs found in sorghum: GA12, GA53, GA44, GA19, GA20, GA1, and GA8. In contrast, the inhibitors that block the conversion of GA20→ GA1, (CGA 163′935 and BX-112) drastically reduced GA1 and GA8 levels, but they either did not change or caused accumulation of intermediates from GA12 to GA20. Combinations of pre-GA12 inhibitors and GA3 plus GA1 strongly reduced GAs other than GA1 and GA3. Each of these compounds inhibited shoot growth in both cultivars and delayed floral initiation in 90M. Floral initiation of 58M was also delayed by CCC, uniconazol, and ancymidol but not by CGA 163`935 and BX-112. This separation of shoot elongation from floral initiation in sorghum is novel. Both inhibition of shoot growth and delayed floral initiation were almost completely relieved by a mixture of GA3 and GA1 in both 58M and 90M. This observation, plus the much lower levels of endogenous GA3 than of GA1 observed in these experiments, implies that GA1 is the major endogenous GA active in shoot elongation. CGA 163′935 and BX-112 also failed to promote tillering in 58M, whereas inhibitors active before GA12 did so. The possibility that the GA20→ GA1 inhibitors fail to block flowering and promote tillering in 58M because biosynthetic intermediates between GA12 and GA20 accumulate and/or because 58M is altered in GA metabolism in this same region of the biosynthetic pathway is discussed.

Location of ethylene production in cotton flowers and dehiscing fruits.

SummaryOver half of the ethylene produced by 1-day-old cotton flowers came from the combined stigma, style, and stamens. These tissues produced 0.0050 μl/flower·h compared to 0.0024 and 0.0010 μl/flower·h produced by the petals and ovary, respectively. Walls of dehiscing cotton fruits produced 0.052 μl ethylene/fruit·h. This was approximately 50% more than seeds plus fiber which produced 0.033 μl/fruit·h.