direct-agroinoculation-maize-seedlings-injection-with-recombinant

Direct Agroinoculation of Maize Seedlings by Injection with Recombinant Foxtail Mosaic Virus and Sugarcane Mosaic Virus Infectious Clones

Agrobacterium-based inoculation approaches are widely used for introducing viral vectors into plant tissues. This study details a protocol for the ...

Iowa State University

Functions of isoamylase-type starch-debranching enzyme (ISA) proteins and complexes in maize (Zea mays) endosperm were characterized. Wild-type endosperm contained three high molecular mass ISA complexes resolved by gel permeation chromatography and native-polyacrylamide gel electrophoresis. Two complexes of approximately 400 kD contained both ISA1 and ISA2, and an approximately 300-kD complex contained ISA1 but not ISA2. Novel mutations of sugary1 (su1) and isa2, coding for ISA1 and ISA2, respectively, were used to develop one maize line with ISA1 homomer but lacking heteromeric ISA and a second line with one form of ISA1/ISA2 heteromer but no homomeric enzyme. The mutations were su1-P, which caused an amino acid substitution in ISA1, and isa2-339, which was caused by transposon insertion and conditioned loss of ISA2. In agreement with the protein compositions, all three ISA complexes were missing in an ISA1-null line, whereas only the two higher molecular mass forms were absent in the ISA2-null line. Both su1-P and isa2-339 conditioned near-normal starch characteristics, in contrast to ISA-null lines, indicating that either homomeric or heteromeric ISA is competent for starch biosynthesis. The homomer-only line had smaller, more numerous granules. Thus, a function of heteromeric ISA not compensated for by homomeric enzyme affects granule initiation or growth, which may explain evolutionary selection for ISA2. ISA1 was required for the accumulation of ISA2, which is regulated posttranscriptionally. Quantitative polymerase chain reaction showed that the ISA1 transcript level was elevated in tissues where starch is synthesized and low during starch degradation, whereas ISA2 transcript was relatively abundant during periods of either starch biosynthesis or catabolism.

Functions of heteromeric and homomeric isoamylase-type starch-debranching enzymes in developing maize endosperm.

Maize  () mutations are beneficial for endosperm nutritional quality but cause negative pleiotropic effects for reasons that are not fully understood. Direct targets of the bZIP transcriptional regulator encoded by  include  and  that specify pyruvate phosphate dikinase (PPDK). This enzyme reversibly converts AMP, pyrophosphate, and phosphoenolpyruvate to ATP, orthophosphate, and pyruvate and provides diverse functions in plants. This study addressed PPDK function in maize starchy endosperm where it is highly abundant during grain fill.  and  were inactivated individually by transposon insertions, and both genes were simultaneously targeted by endosperm-specific RNAi.  accounts for the large majority of endosperm PPDK, whereas  specifies the abundant mesophyll form. The  mutation is seedling-lethal, indicating that C4 photosynthesis is essential in maize. RNAi expression in transgenic endosperm eliminated detectable PPDK protein and enzyme activity. Transgenic kernels weighed the same on average as nontransgenic siblings, with normal endosperm starch and total N contents, indicating that PPDK is not required for net storage compound synthesis. An opaque phenotype resulted from complete PPDK knockout, including loss of vitreous endosperm character similar to the phenotype conditioned by -. Concentrations of multiple glycolytic intermediates were elevated in transgenic endosperm, energy charge was altered, and starch granules were more numerous but smaller on average than normal. The data indicate that PPDK modulates endosperm metabolism, potentially through reversible adjustments to energy charge, and reveal that - mutations can affect the opaque phenotype through regulation of PPDK in addition to their previously demonstrated effects on storage protein gene expression.

Ryan R. Lappe

Department of Plant Pathology and Microbiology

Iowa State University

Direct Agroinoculation of Maize Seedlings by Injection with Recombinant Foxtail Mosaic Virus and Sugarcane Mosaic Virus Infectious Clones

Ryan R. Lappe

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Plant Pathology and Microbiology

Iowa State University

Direct Agroinoculation of Maize Seedlings by Injection with Recombinant Foxtail Mosaic Virus and Sugarcane Mosaic Virus Infectious Clones

Department of Plant Pathology and Microbiology