Maize, the highest-yielding cereal crop worldwide, is particularly susceptible to drought during its 2- to 3-week flowering period. Many genetic engineering strategies for drought tolerance impinge on plant development, reduce maximum yield potential or do not translate from laboratory conditions to the field. We overexpressed a gene encoding a rice trehalose-6-phosphate phosphatase (TPP) in developing maize ears using a floral promoter. This reduced the concentration of trehalose-6-phosphate (T6P), a sugar signal that regulates growth and development, and increased the concentration of sucrose in ear spikelets. Overexpression of TPP increased both kernel set and harvest index. Field data at several sites and over multiple seasons showed that the engineered trait improved yields from 9% to 49% under non-drought or mild drought conditions, and from 31% to 123% under more severe drought conditions, relative to yields from nontransgenic controls.

Plant peroxidases play a major role in lignin formation and wound healing and are believed to be involved in auxin catabolism and defense to pathogen attack. The function of the anionic peroxidase isozymes is best understood in tobacco. These isozymes catalyze the formation of the lignin polymer and form rigid cross-links between lignin, cellulose, and extensin in the secondary plant cell wall. We report the purification of the anionic peroxidase isozymes from tobacco and their partial amino acid sequence.

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ABSTRACrPeroxidases (EC 1.11.1.7) have been implicated in the responses of plants to physical stress and to pathogens, as well as in a variety of cellular processes including cell wall biosynthesis. Tissue samples from leaf, root, pith, and callus of Nicotiama tabacum were assayed for specific peroxidase isozymes by analytical isoelectric focusing. Each Xanthi plants were grown from seed in the greenhouse with 14-h daily light periods. Tobacco callus cultures were derived from sections ofpith from N. tabacum L. cv Xanthi. Callus was grown on agar plates and subcultured every 14 d. The medium contained Murashige-Skoog salts, B5 vitamins, 2% sucrose, 0.4 mg/ L 2,4-D, 0.05 mg/L kinetin, and 0.8% agar, and callus was grown in fluorescent light at 28°C (6).Wounding and TMV' Infection. Tobacco leaves were wounded by crushing with a hemostat at a 45°angle to the midvein. Pith was wounded by cutting out 5-mm wedges from the stem at 5-cm intervals or by aseptically removing the pith with a cork borer and slicing into 3-mm cross-sections. These sections were incubated for various times in a moist dark chamber either in water alone or water containing 5 jug/ml cycloheximide. In all cases tissue samples were combined from three or more mature healthy plants. All experiments were repeated at least three times with similar results.Eight-week-old Xanthi plants were infected with TMV by gently rubbing the leaves with carborundum and then applying an aqueous virus suspension with a cotton applicator. Approximately 50 necrotic lesions were detected 2 d after infection. Excessive wounding of the leaves was avoided.Extraction Procedure, Protein Assay, and Enzyme Assay. Tissue samples were homogenized on ice with a Polytron homogenizer (Brinkman Industries) at setting 8 for 20 s in 10 mm sodium phosphate buffer (pH 6.0). The ratios of buffer volume to tissue mass were as follows: 3 to 1, for leaftissue; 3 to 1 for root tissue; 2 to 1 for pith tissue; and 1 to 1 for callus tissue. The homogenate was centrifuged for 20 min at 15,000g, and the cleared supernate was saved for analysis. Protein content was determined by the Bradford reagent method (BioRad). Peroxidase activity was assayed in 0.28% guaiacol, 0.05 M sodium phosphate buffer (pH 6.0), and 0.3% H202. The increase in absorbance was monitored at 470 nm in a Gilford spectrophotometer. Samples prepared for isoelectric focusing included 1% PVP to decrease binding by phenolic compounds which may affect the number of isozymes (21).Flat Bed Isoelectric Focusing and in Situ Peroxidase Staining. Tissue extracts were subjected to analytical flat bed isoelectric focusing on polyacrylamide gels containing ampholines in the pH range 3.5 to 9.5 (LKB, Bromma, Sweden). The samples were subjected to electrophoresis for 1.5 h at 0.125 W/cm2 at 10°C. After focusing, the gels were soaked for 30 min in 500 ml of PBS (10 mm sodium phosphate buffer [pH 6.0], 150 mM NaCl) to remove the ampholines and equalize the pH throughout the gel. The isozymes at the extremes ofthe pH gradient would otherwise ...

We have studied the mechanism of UV protection in two duckweed species (Lemnaceae) by exploiting the UV sensitivity of photosystem II as an in situ sensor for radiation stress. A UV-tolerant Spirodela punctata G.F.W. Meyer ecotype had significantly higher indole-3-acetic acid (IAA) levels than a UV-sensitive ecotype. Parallel work on Lemna gibba mutants suggested that UV tolerance is linked to IAA degradation rather than to levels of free or conjugated IAA. This linkage is consistent with a role for class III phenolic peroxidases, which have been implicated both in the degradation of IAA and the cross-linking of various UV-absorbing phenolics. Biochemical analysis revealed increased activity of a specific peroxidase isozyme in both UV-tolerant duckweed lines. The hypothesis that peroxidases play a role in UV protection was tested in a direct manner using genetically modified tobacco (Nicotiana sylvestris). It was found that increased activity of the anionic peroxidase correlated with increased tolerance to UV radiation as well as decreased levels of free auxin. We conclude that phenol-oxidizing peroxidases concurrently contribute to UV protection as well as the control of leaf and plant architecture.

SummaryExtended darkness induces a transient increase in sugars and trehalose pathway gene expression.

Indole-3-acetic acid (IAA) is a powerful plant growth regulator. The oxidative decarboxylation of IAA by plant peroxidases is thought to be a major degradation reaction involved in controlling the in vivo level of IAA. Horseradish peroxidase isoenzyme C and an anionic tobacco peroxidase isolated from transgenic Nicotiana sylvestris have been used in experiments in vitro designed to determine the mechanism of IAA oxidation. In particular, the initial reduction of ferric to ferrous enzyme, a key step in previously proposed mechanisms, has been investigated by rapid-scan stopped-flow spectrophotometry under strictly anaerobic conditions and at defined oxygen concentrations. The data provide the first evidence for a ternary complex comprising peroxidase, IAA and oxygen that is kinetically competent both at the initiation stage and during the catalytic cycle of IAA oxidation. A general scheme describing the oxidative cycles of both anionic and cationic peroxidases is proposed that includes native ferric enzyme and compound II as kinetically competent intermediates. For anionic peroxidases, addition of hydrogen peroxide switches on the oxidative cycle thereby promoting IAA oxidation. 2-Methyl-IAA is not a substrate of the oxidase reaction, suggesting a specific interaction between plant peroxidases and IAA.

Little is known about how salt impacts primary metabolic pathways of C 4 plants, particularly related to kernel development and seed set. Osmotic stress was applied to maize (Zea mays) B73 by irrigation with increasing concentrations of NaCl from the initiation of floral organs until 3 d after pollination. At silking, photosynthesis was reduced to only 2% of control plants. Salt treatment was found to reduce spikelet growth, silk growth, and kernel set. Osmotic stress resulted in higher concentrations of sucrose (Suc) and hexose sugars in leaf, cob, and kernels at silking, pollination, and 3 d after pollination. Citric acid cycle intermediates were lower in salt-treated tissues, indicating that these sugars were unavailable for use in respiration. The sugar-signaling metabolite trehalose-6-phosphate was elevated in leaf, cob, and kernels at silking as a consequence of salt treatment but decreased thereafter even as Suc levels continued to rise. Interestingly, the transcripts of trehalose pathway genes were most affected by salt treatment in leaf tissue. On the other hand, transcripts of the SUCROSE NONFERMENTING-RELATED KINASE1 (SnRK1) marker genes were most affected in reproductive tissue. Overall, both source and sink strength are reduced by salt, and the data indicate that trehalose-6-phosphate and SnRK1 may have different roles in source and sink tissues. Kernel abortion resulting from osmotic stress is not from a lack of carbohydrate reserves but from the inability to utilize these energy reserves.