Classical arabinogalactan proteins (AGPs) are an abundant class of cell surface proteoglycans widely distributed in flowering plants. We have used a combination of enhancer detection tagging and RNA interference (RNAi)-induced posttrancriptional silencing to demonstrate that AGP18, a gene encoding a classical arabinogalactan protein, is essential for female gametogenesis in Arabidopsis thaliana. AGP18 is expressed in cells that spatially and temporally define the sporophytic to gametophytic transition and during early stages of seed development. More than 75% of the T1 transformants resulted in T2 lines showing reduced seed set during at least three consecutive generations but no additional developmental defects. AGP18-silenced T2 lines showed reduced AGP18 transcript levels in female reproductive organs, the presence of 21-bp RNA fragments specific to the AGP18 gene, and the absence of in situ AGP18 mRNA localization in developing ovules. Reciprocal crosses to wild-type plants indicate that the defect is female specific. The genetic and molecular analysis of AGP18-silenced plants containing a single T-DNA RNAi insertion suggests that posttranscriptional silencing of AGP18 is acting both at the sporophytic and gametophytic levels. A cytological analysis of all defective AGP18-RNAi lines, combined with the analysis of molecular markers acting at key stages of female gametogenesis, showed that the functional megaspore fails to enlarge and mitotically divide, indicating that AGP18 is essential to initiate female gametogenesis in Arabidopsis. Our results assign a specific function in plant development to a gene encoding a classical AGP.
Late embryogenesis abundant (LEA) proteins are a large protein family that mainly function in protecting cells from abiotic stress, but these proteins are also involved in regulating plant growth and development. In this study, we performed a functional analysis of LEA13 and LEA30 from Arabidopsis thaliana. The results showed that the expression of both genes increased when plants were subjected to drought-stressed conditions. The insertional lines lea13 and lea30 were identified for each gene, and both had a T-DNA element in the regulatory region, which caused the genes to be downregulated. Moreover, lea13 and lea30 were more sensitive to drought stress due to their higher transpiration and stomatal spacing. Microarray analysis of the lea13 background showed that genes involved in hormone signaling, stomatal development, and abiotic stress responses were misregulated. Our results showed that LEA proteins are involved in drought tolerance and participate in stomatal density.
Although more than 25,000 genes of Arabidopsis thaliana have been sequenced and mapped, adequate expression or functional information is available for less than 15% of them. In the case of Oryza sativa (rice), about half of more than 55,000 predicted genes have been assigned to a vague functional category on the basis of their sequence, but fewer than 100 have been ascribed a precise, verified function after the identification of a mutant phenotype caused by the molecular disruption of the corresponding gene. Enhancer detection and gene trapping represent insertional mutagenesis strategies that report random expression of many genes and often generate loss-of-function mutations. Several trapping vectors have been designed in a limited number of species, and large-scale enhancer detection and gene trap screens that aim to generate a wide range of spatially and temporally restricted expression patterns have been initiated in both Arabidopsis and rice. These strategies are proving to be essential to the functional annotation of completely sequenced genomes, enabling the analysis of gene function in the context of the entire plant life cycle and substantially expanding our understanding of plant growth and development.
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