The Arabidopsis group 1 LATE EMBRYOGENESIS ABUNDANT protein ATEM6 is required for normal seed development

Abstract

As part of the embryo maturation process, orthodox seeds undergo a developmentally regulated dehydration period. The LATE EMBRYOGENESIS ABUNDANT (LEA) genes encode a large and diverse family of proteins expressed during this time. Many hypothesize that LEA proteins act by mitigating water loss and maintaining cellular stability within the desiccated seed, although the mechanisms of their actions remain largely unknown. The model plant Arabidopsis (Arabidopsis thaliana) contains two genes belonging to the group 1 LEA family, ATEM1 and ATEM6, and knockout mutations in these genes are being sought as a means to better understand group 1 LEA protein function during embryo maturation. We have identified a T-DNA insertion allele of the ATEM6 gene in which the T-DNA is present just downstream of the protein coding region. While this gene is transcriptionally active and encodes a wild-type protein, there is no detectable ATEM6 protein in mature seeds. Mutant seeds display premature seed dehydration and maturation at the distal end of siliques, demonstrating that this protein is required for normal seed development. We propose that one function for group 1 LEA proteins in seed development is to buffer the water loss that occurs during embryo maturation and that loss of ATEM6 expression results in the mutant phenotype.

Figure 1.

Schematic of pCSA110 T-DNA in ATEM6. The T-DNA region is shown as a simple, single insertion in ATEM6 genomic DNA (A) and the actual structure as determined by plasmid rescue and sequence analysis of the RB/ATEM6 and LB/ATEM6 junctions (B). Genomic DNA is black. The T-DNA, bounded by the RB and LB sequences (hatched), is gray, and the pBS portion of the T-DNA is stippled. Exons of the ATEM6 gene are shown as thick bars (I, II, and 3′-UTR). Note that the 3′-UTR region of exon II is physically separated from the remainder of the gene by the T-DNA insertion. Deletions of T-DNA identified by sequence analysis are underlined in A and absent in B. Restriction enzyme sites for EcoRI (E), NcoI (Nc), and BamHI (B) are indicated.

Figure 2.

Southern-blot analysis of individual 6.413 mutant plants. A 10-μg sample of genomic DNA was digested with BamHI, fragmented on a 0.7% agarose gel, transferred to nylon membrane, and hybridized with an ATEM6 gene-specific 3′-UTR probe. WT, Wild type; numbers indicate laboratory plant line numbers. Molecular marker sizes are indicated to the left.

Figure 3.

Mapping of chromosome 2. Schematic of map positions of the Thy1, NGA168, and ML markers and the ATEM6 locus. The genetic distance was derived from recombination analysis of 100 individual F₂ plants. Distances are given as percentage recombination.

Figure 4.

RT-PCR of wild-type and atem6-1 RNA. Total RNA extracted from 10 seeds per sample was reverse transcribed using a tailed oligo(dT) primer and subsequently subjected to PCR using ATEM6 exon-specific primers. A portion of the RT-PCR reaction was run on a 1.2% agarose gel. Note this is not a quantitative measure of mRNA levels. Lane 1, RNA markers (sizes shown to the left); lane 2, wild type; lane 3, atem6-1 mutant; lane 4, ATEM6 genomic clone; and lanes 5 and 6, no RT controls for wild type and atem6-1 mutants, respectively.

Figure 5.

RNA gel-blot analysis of wild-type and atem6-1 RNA. Total RNA (10 μg) from mature seeds was separated on a 1.2% agarose formaldehyde gel and transferred to nylon membrane (A) and hybridized with an ATEM6 gene-specific 5′-UTR probe (B). Lane 1, atem6-1 mutant; lane 2, wild type. RNA M_r marker sizes are indicated to the left.

Figure 6.

Immunoblot analysis of wild-type and atem6-1 protein. Total protein extracted from seeds from single siliques was separated on a 15% SDS-polyacrylamide gel and transferred to PVDF membrane. ATEM proteins were detected with an anti-wheat Em protein antibody. Note this is not a quantitative measure of protein levels. Lane 1, atem6-1 mutant; lane 2, ATEM6/atem6-1 heterozygote; lane 3, wild type. ATEM1 and ATEM6 proteins are indicated to the left.

Figure 7.

Phenotype of wild-type (A), atem6-1 mutant (B), and ATEM6-C (complementation; C) seeds and siliques. Siliques were removed from the plants at 13 daf and examined under a dissecting microscope. For each genotype, top image represents a whole silique; middle image is the same silique after opening; and bottom image is a closer view of the plant proximal (left) and plant distal (right) ends of the same silique.