Maize high chlorophyll fluorescent 60 mutation is caused by an Ac disruption of the gene encoding the chloroplast ribosomal small subunit protein 17.

The maize mutation high chlorophyll fluorescence 60-muTable 1 (hcf60-m1), generated through Activator (Ac) tagging, has insufficient photosynthetic electron transport. Here we show that the Hcf60 gene encodes a protein with substantial amino acid similarity to plant plastid and bacterial ribosomal small subunit protein 17 (RPS17) proteins. The lack of detectable HCF60 transcripts in mutant leaves, and insertion of the transposed Ac element 17 bp upstream of the start of translation in the mutated locus, suggest that little if any RPS17 is produced. The mutant phenotype is consistent with reduced plastid translation. Seedling lethal hcf60-m1 plants display temperature and light-dependent chlorophyll deficiencies, a depletion of plastid rRNA pools, and few high-molecular-weight polysomal complexes. Growth under moderate light conditions (27 degrees C, 100 microE m-2 sec-1) allows for substantial chlorophyll accumulation in mutant leaves, yet the number of functional photosystem II complexes appears low. Nevertheless, the presence of a limited but intact C4 system indicates that some plastid translation occurs.

liguleless1 encodes a nuclear-localized protein required for induction of ligules and auricles during maize leaf organogenesis.

The maize ligule and auricle are structures on the maize leaf that develop at the boundary of the sheath and blade. In the absence of liguleless1 (Ig1) gene expression, ligule and auricle are not formed, and the blade-sheath boundary does not develop as an exact line between sheath and blade. By using the Activator (Ac) transposable element as a molecular tag, a novel Ig1 allele, Ig1-m1, was isolated and cloned. Analysis of somatic revertant sectors confirmed that the LG1 gene product functions in a cell-autonomous fashion. cDNA cloning as well as RT-PCR analysis of the LG1 mRNA indicate that the Ig1 gene is expressed at very low levels in the ligular region of developing maize leaf primordia, perhaps as early as plastochron 6 or earlier. Cellular localization studies in a heterologous system indicate that the LG1 product localizes exclusively to the nucleus. The predicted amino acid sequence of the LG1 protein is largely novel but contains an internal domain of 77 amino acids with significant similarity to a domain present in two recently identified SQUAMOSA PROMOTER-BINDING proteins 1 and 2 (SBP1 and SBP2) in Antirhinum majus.

Transformation of Maize Cells and Regeneration of Fertile Transgenic Plants.

A reproducible system for the generation of fertile, transgenic maize plants has been developed. Cells from embryogenic maize suspension cultures were transformed with the bacterial gene bar using microprojectile bombardment. Transformed calli were selected from the suspension cultures using the herbicide bialaphos. Integration of bar and activity of the enzyme phosphinothricin acetyltransferase (PAT) encoded by bar were confirmed in all bialaphos-resistant callus lines. Fertile transformed maize plants (R0) were regenerated, and of 53 progeny (R1) tested, 29 had PAT activity. All PAT-positive progeny analyzed contained bar. Localized application of herbicide to leaves of bar-transformed R0 and R1 plants resulted in no necrosis, confirming functional activity of PAT in the transgenic plants. Cotransformation experiments were performed using a mixture of two plasmids, one encoding PAT and one containing the nonselected gene encoding [beta]-glucuronidase. R0 plants regenerated from co-transformed callus expressed both genes. These results describe and confirm the development of a system for introduction of DNA into maize.

Metabolic Requirement of Cucurbita pepo for Boron.

Lateral roots of intact summer squash seedlings (Cucurbita pepo L.) were used to quantify the effects of boron deficiency on DNA synthesis, protein synthesis, and respiration. The temporal relationship between changes in these metabolic activities and the cessation of root elongation caused by boron deprivation was determined. Transferring 5-day-old squash seedlings to a hydroponic culture medium without boron for 6 hours resulted in a 62% reduction in net root elongation and a 30% decrease in the incorporation of [(3)H]thymidine into DNA by root tips (apical 5-millimeter segments). At this time, root tips from both boron-deficient and boron-sufficient plants exhibited nearly identical rates of incorporation of [(14)C]leucine into protein and respiration as measured by O(2) consumption. After an additional 6 hours of boron deprivation, root elongation had nearly ceased. Concomitantly, DNA synthesis in root apices was 66% less than in the boron-sufficient control plants and protein synthesis was reduced 43%. O(2) consumption remained the same for both treatments. The decline and eventual cessation of root elongation correlated temporally with the decrease in DNA synthesis, but preceded changes in protein synthesis and respiration. These results suggest that boron is required for continued DNA synthesis and cell division in root meristems.

Recovery of a soybean urease genomic clone by sequential library screening with two synthetic oligodeoxynucleotides.

We report the first isolation of a low-copy-number gene from a complex higher plant (soybean) genome by direct screening with synthetic oligodeoxynucleotide (oligo) probes. A synthetic, mixed, 21-nucleotide (nt) oligo (21-1) based on a seven amino acid (aa) sequence from soybean seed urease, was used to screen genomic libraries of soybean (Glycine max [L.] Merr.) in the lambda Charon 4 vector. Twenty homologous clones were recovered from a screen of 500,000 plaques. These were counterscreened with embryo-specific cDNA (15-2 cDNA) made by priming with a second, mixed 15-nt oligo (15-2), based on a Jack bean (Canavalia ensiformis) urease peptide [Takishima et al., J. Natl. Def. Med. Coll. 5 (1980) 19-23]. Five out of 20 clones were homologous to 15-2 cDNA and proved to be identical. Nucleotide sequence analysis of representative clone E15 confirmed that it contained urease sequences. Subclones of E15 homologous to the oligo probes contain a deduced amino acid sequence which matches 108 of 130 aa residues of an amino acid run in a recently published [Mamiya et al., Proc. Jap. Acad. 61B (1985) 359-398] complete protein sequence for Jack-bean seed urease. Using clone E15 as a probe of soybean embryonic mRNA revealed a homologous 3.8-kb species that is the size of the urease messenger. This species is absent from mRNA of embryos of a soybean seed urease-null mutant. However, both urease-positive and urease-null genomes contain the 11-kb DNA fragment bearing urease sequences.

Structure and possible ureide degrading function of the ubiquitous urease of soybean.

Ubiquitous soybean urease, as opposed to the seed-specific urease, designates the seemingly identical ureolytic activities of suspension cultures and leaves. It also appears to be the basal urease in developing seeds of a variety, Itachi, which lacks the seed-specific urease (Polacco, Winkler 1984 Plant Physiol 74: 800-804). On native polyacrylamide gels the ureolytic activities in crude extracts of these three tissues comigrate as determined by assays of gel slices. At this level of resolution the ubiquitous urease also migrates with or close to the fast (trimeric) form of the seed-specific urease.The ubiquitous urease was purified approximately 100-fold from suspension cultures of two cultivars (Itachi and Prize) as well as from developing seeds of Itachi. These partially purified preparations allowed visualization of native urease on polyacrylamide gels by activity staining and of urease subunits on denaturing lithium dodecyl sulfate gels by electrophoretic transfer to nitrocellulose and immunological detection ("Western Blot"). The ubiquitous urease holoenzyme migrates slightly less rapidly than the fast seed urease in native gels; its subunit migrates slightly less rapidly than the 93.5 kilodaltons subunit of either the fast or slow (hexameric) seed enzyme. The ubiquitous urease elutes from an agarose A-0.5 meter column with the fast form of the seed urease species suggesting that the ubiquitous urease, like the fast seed urease, exists as a trimeric holoenzyme. The soybean cultivar, Prize, produces the hexameric seed urease; yet its ubiquitous urease (from leaf and suspension culture) is trimeric.The pH dependence of the ureolytic activity of seed coats of both seed urease-negative (Itachi) and seed urease-positive (Williams) cultivars suggests that this activity is exclusively the ubiquitous urease. Its relatively higher levels in seed coats than in embryos of Itachi suggests that the ubiquitous urease is involved in degradation of urea derived from ureides. Consistent with a ureide origin for urea is the observation that addition of a urease inhibitor, phenylphosphordiamidate, to extracts of developing Itachi seeds (seed coat plus embryo) results in accumulation of urea from allantoic acid.

Photosynthesis in Tall Fescue : V. Analysis of High PSI Activity in a Decaploid Genotype.

Previous work in our laboratory (Krueger, Miles 1981 Plant Physiol 68: 1110-1114) indicated that a decaploid genotype (I-16-2) of tall fescue (Festuca arundinacea Schreb.) which exhibits unusually high net photosynthesis rates also had high potential rates of photosynthetic electron transport through photosystem I (PSI) compared to the typical hexaploid genotype (V6-802). Analysis of electron transport activity revealed that the oxidizing side of PSI as the major site of difference. Examination of the whole thylakoids and subchloroplast particle protein components of the common hexaploid and the decaploid genotypes had major polypeptide differences at 30, 21, and 12.5 kilodaltons. These differences could not be assigned to a specific physiological function in PSI. The decaploid had increased P(700) and plastocyanin content on a chlorophyll basis. Antibodies raised against fescue plastocyanin were used to quantitate plastocyanin in crude (Triton X-100) solubilized extracts of plant material. Results showed that the decaploid had 16% and 40% more plastocyanin on a weight and area basis, respectively. The antibodies did not inhibit electron transport (diaminodiurene to methyl viologen) in isolated thylakoids strengthening the hypothesis of plastocyanin as an internal mobile electron shuttle. The trend of inhibition of plastocyanin by KCN was similar in the two genotypes but the decaploid had 15 to 20% higher rates of electron flow under nearly all inhibiting conditions.

Chlorophyll-Protein Complexes of a Photosystem II Mutant of Maize : Evidence that Chlorophyll-Protein a-2 and a Chlorophyll-Protein Complex Derived from a Photosystem I Antennae System Comigrate on Polyacrylamide Gels.

Use of the octyl beta-d-glucopyranoside solubilization procedure of Camm and Green (1980 Plant Physiol 66: 428-432) reveals that thylakoid membranes of a photosystem (PS) II-deficient maize (Zea mays L.) mutant lack two chlorophyll protein (CP) complexes associated with PSII, i.e. CPa-1 and CPa-2. In contrast, when lithium dodecyl sulfate is used to solubilize the membranes of the mutant prior to electrophoretic separation, a CP complex is observed which has a mobility similar to that of CPa-2. Comparison of spectral characteristics and polypeptide composition of the green bands in this region taken from samples of the mutant, normal sibling control plants and from PSII preparations indicate that the CP complex observed in the mutant represents a portion of a light-harvesting complex of PSI (Mullet et al. 1980 Plant Physiol 65: 814-822). The green band observed in normal maize samples can contain both the CPa-2 complex as well as the CP complex derived from the PSI antennae system.

Photosynthesis in Fescue : III. RATES OF ELECTRON TRANSPORT IN A POLYPLOID SERIES OF TALL FESCUE PLANTS.

Photosystem I electron transport activity has been found to be considerably higher in a decaploid tall fescue (Festuca arundinacea Schreb.) genotype as compared to a common hexaploid genotype. The decaploid genotype also displayed a higher photosystem whole chain (Photosystem II plus Photosystem I uncoupled) activity, suggesting a connection between polyploidy and increased electron transport activity. However, when a polyploidy series of tall fescue, ranging from diploid to decaploid with several different genetic isolates at each ploidy level, was examined in natural growth conditions, no effect of increasing genome content on electron transport and photophosphorylation was found. These results suggest that a gene component of one of the genomes involved may be responsible for the increased activity rather than simply the total chromosome content.

Photosynthesis in Fescue: I. HIGH RATE OF ELECTRON TRANSPORT AND PHOSPHORYLATION IN CHLOROPLASTS OF HEXAPLOID PLANTS.

Chloroplasts isolated from tall fescue, Festuca arundinacea Schreb., showed high rates of electron transport, comparable to rates observed for spinach chloroplasts.Chloroplasts were well coupled and rates of electron transport from water to methyl viologen (photosystem II and I) were increased two to five times when ADP and inorganic phosphate or methylamine (uncoupler) were added to the reaction mixture. Ratios of P:2e for photosystem II plus I were found to be near 1.2. Electron transport rates from water to p-phenylenediamine or 2,6-dichlorobenzoquinone (photosystem II) were over 300 micromoles O(2) per hour per milligram chlorophyll, while P:2e ratios were found to be over 0.5. The highest rates of electron transport were found in electron flow from diaminodurene to methyl viologen (photosystem I) and P:2e ratios remained near 0.5.Light intensity saturation curves for photosystem II and I, as well as the photosystems independently, resembled curves for spinach, with saturation of electron transport in photosystem I and photosystem II separately occurring at 35% of the available light intensity (6000 microeinsteins per square meter per second). Photosystem II and I in sequence were saturated at about half this light intensity.