Molecular Duckweed: 
Duckweed Mutants

alpha-Methyltryptophan-Resistant Mutants and their Auxin Levels
Tam, Y.Y., Cohen, J.D., Slovin, J.P.
Selection and characterization of alpha-methyltryptophan-resistant lines of Lemna gibba showing a rapid rate of indole-3-acetic acid turnover.
Plant Physiology (Lancaster, Pa.) Jan 1995. v. 107 (1)p. 77-85.

Turnover rate is an important aspect of the regulation of plant processes by plant growth substances. To study turnover of indole-3-acetic acid (IAA), two alpha-methyltryptophan-resistant lines (MTR1 and MTR2) of Lemna gibba were generated by nitrosomethyl urea treatment of an inbred line derived from L gibba G-3. In this report we describe: (a) the development of a selection system using this near isogenic line of L gibba; (b) techniques for chemical mutation of the lines and selection for a-methyltryptophan resistance; and (c) the partial characterization of the selected lines. MTR lines contained 3-fold higher levels of anthranilate synthase activity. The enzyme in the MTR lines required higher levels of tryptophan for feedback inhibition. MTR lines also contained 8-fold higher levels of  tryptophan, 3-fold higher levels of free IAA, and similar levels of total IAA compared to the inbred line. Turnover rates in the inbred and selected lines were calculated, using the first-order rate equation, based on the decrease over time in isotopic enrichment of l3C6-IAA introduced into L gibba during a 1-h pulse period. Isotope enrichment in IAA was determined by using gas chromatography-mass spectrometry. Both MTR lines had an approximately 1 O-fold higher rate of IAA turnover than the parent inbred line.

Slovin, J.P., Cohen, J.D.
Levels of indole-3-acetic acid in Lemna gibba G-3 and in a large Lemna mutant regenerated from tissue culture.
Plant Physiology. Feb 1988. v. 86 (2) p. 522-526. ill.

A Mutant in Photosynthesis
Bruce, B.D., Malkin, R.
Biosynthesis of the chloroplast cytochrome b6f complex: studies in a photosynthetic mutant of Lemna.
The Plant Cell. Feb 1991. v. 3 (2) p. 203-212. ill.

The biosynthesis of the cytochrome b6f complex has been studied in a mutant, no. 1073, of Lemna perpusilla that contained less than 1% of the four protein subunits when compared with a wild-type strain. RNA gel blot analyses of the mutant indicated that the chloroplast genes for cytochrome f, cytochrome b6, and subunit IV (petA, petB, and petD, respectively) are transcribed and that the petB and petD transcripts undergo their normal processing. Analysis of polysomal polyA+ RNA indicated that the level of translationally active mRNA for the nuclear-encoded Rieske Fe-S protein (petC) was reduced by >100-fold in the mutant. Immunoprecipitation of in vivo labeled proteins indicated that both cytochrome f and subunit IV are synthesized and that subunit IV has a 10-fold higher rate of protein turnover in the mutant. These results are discussed in terms of the assembly of the cytochrome complex and the key role of the Rieske Fe-S protein in this process.  

Vener, A.V., Kan, P.J.M. van.,  Gal, A., Andersson, B., Ohad, I.
Activation/deactivation cycle of redox-controlled thylakoid protein phosphorylation: role of plastoquinol bound to the reduced cytochrome bf  complex.
The Journal of Biological Chemistry. Oct 20, 1995. v. 270 (42) p. 25225-25232.
Signal transduction via light-dependent redox control of reversible thylakoid protein phosphorylation has evolved in plants as a unique mechanism for controlling events related to light energy utilization. Here we report for the first time that protein phosphorylation can be activated without light or the addition of reducing agents by a transient exposure of isolated thylakoid membranes to low pH in darkness. The activation of the kinase after incubation of dark-adapted thylakoids at pH 4.3 coincides with an increase in the plastoquinol: plastoquinone ratio up to 0.25. However, rapid plastoquinol reoxidation (< 1 min) at pH 7.4 contrasts with the slow kinase deactivation (t1/2 = 4 min), which indicates that the redox control is not directly dependent on the plastoquinone pool. Use of inhibitors and a cytochrome bf-deficient mutant of Lemna demonstrate
the involvement of the cytochrome bf complex in the low-pH induced protein phosphorylation. EPR spectroscopy shows that subsequent to the transient low pH treatment and transfer of the thylakoids to pH 7.4, cytochrome f, the Rieske Fe-S center, and plastocyanin become reduced and are not reoxidized while the kinase is slowly deactivated. However, the deactivation correlates with a decrease of the EPR gz signal of the reduced Rieske Fe-S center, which is also affected by quinone analogues that inhibit the kinase. Our data point to an activation mechanism of thylakoid protein phosphorylation that involves the binding of plastoquinol to the cytochrome bf complex in the vicinity of the reduced Rieske Fe-S center.

Gower, R.A., Posner, H.B.
Effects of light and 3-(3,4-dichlorophenyl)-1,1-dimethylurea on levels of ATP in Lemna paucicostata 6746 and a photosynthetic mutant with abnormal flowering responses.
Plant Physiology Mar 1979. v. 63 (3)  p. 548-551. ill.

Witztum, A., Posner, H.B., Gower, R.A.
Phototactic chloroplast displacement in the photosynthetic mutant, Lemna paucicostata strain 1073.
Annals of Botany. July 1979. v. 44 (1)
p. 1-4. ill., plate.

Chloroplast Genes
Jordan, W.C., Courtney, M.W. , Neigel, J.E.
 Low levels of intraspecific genetic variation at a rapidly evolving chloroplast DNA locus in North American duckweeds (Lemnaceae).
American Journal of Botany. Apr 1996. v. 83 (4) p. 430-439.
Although most previous studies on chloroplast (cp) DNA variation in plants have concentrated on systematics and evolution above the species level, intraspecific variation in cpDNA is common and has provided useful insights into population-level evolutionary processes. Polymerase chain reaction methods were used to examine restriction site and sequence variation in the chloroplast rpL16 gene within and among populations of duckweed species (Spirodela and Lemna) from the southern and eastern United States. To our knowledge, the rpL16 region has not previously been used to investigate cpDNA variation in nature. While considerable restriction site and sequence variation were detected among species, no variation was found within populations of either of the two species (S. punctata and L. minor) selected for sequence analysis, and S. punctata showed no interpopulational variation. Two cpDNA haplotypes were identified in L. minor, with one haplotype restricted to three sites in Louisiana and the other found in all other populations sampled. This paucity of variation cannot be readily explained as the result of a low mutation rate. In general, group II introns appear to be subject to very little functional constraint, and extensive sequence differences have been found between species in the chloroplast rpL16 intron in particular. However, factors such as historical range expansions and contractions, founding effects, fluctuations in local population size, and natural selection may play a role in reducing cpDNA sequence variability in these species.
NPR1 Gene
Weatherwax, S.C.,  Tingay, S., Tobin, E.M.
The phytochrome response of the Lemna gibba NPR1 gene is mediated primarily through changes in abscisic acid levels.
Plant Physiology (Lancaster, Pa.) Apr 1998. v. 116 (4) p. 1299-1305.
Two important signaling systems involved in the growth and development of plants, those triggered by the photoreceptor phytochrome and the hormone abscisic acid (ABA), are involved in the regulation of expression of the NPR1 gene of Lemna gibba. We previously demonstrated that phytochrome action mediates changes in ABA levels in L. gibba, correlating with changes in gene expression evoked by stimulation of the phytochrome system. We have now further characterized phytochrome- and ABA-mediated regulation of L. gibba NPR1 gene expression using a transient particle bombardment assay, demonstrating that regulatory elements controlling responses to both stimuli reside within 156 nucleotides upstream of the transcription start. Linker scan (LS) analysis of the region from -156 to -70 was used to identify two specific requisite and nonredundant cis-acting promoter elements between -143 to -135 (LS2) and -113 to -101 (LS5). Mutation of either of these elements resulted in a coordinate loss of regulation by phytochrome and ABA. This suggests that, unlike the L. gibba Lhcb2*1 promoter, in which phytochrome and ABA regulatory elements are separable, the phytochrome response of the L. gibba NPR1 gene can be attributed to alterations in ABA levels.

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Revised:  May 6, 2004