sal1 determines the number of aleurone cell layers in maize endosperm and encodes a class E vacuolar sorting protein

Abstract

A microscopy-based screen of a large collection of maize Mutator (Mu) transposon lines identified the supernumerary aleurone layers 1-1 (sal1-1) mutant line carrying up to seven layers of aleurone cells in defective kernel endosperm compared with only a single layer in wild-type grains. Normal, well filled endosperm that is homozygous for the sal1-1 mutant allele contains two to three layers of aleurone cells. Cloning of the sal1 gene was accomplished by using Mu tagging, and the identity of the cloned gene was confirmed by isolating an independent sal1-2 allele by reverse genetics. Homozygous sal1-2 endosperm has two to three layers of aleurone cells in normal, well filled grains. In situ hybridization experiments reveal that the sal1 gene is ubiquitously expressed in vegetative as well as zygotic grain tissues, with no difference being detected between aleurone cells and starchy endosperm cells. Northern blot analysis failed to detect the sal1-2 transcript in leaves of homozygous plants, suggesting that the allele is a true sal1 knockout allele. The sal1 gene encodes a homologue of the human Chmp1 gene, a member of the conserved family of the class E vacuolar protein sorting genes implicated in membrane vesicle trafficking. In mammals, CHMP1 functions in the pathway targeting plasma membrane receptors and ligands to lysosomes for proteolytic degradation. Possible roles for the function of the sal1 gene in aleurone signaling, including a defect in endosome trafficking, are discussed.

Fig. 1.

The phenotype of sal1 endosperm and vegetative plant tissues. (A) Homozygous sal1-1 defective kernels. Two wild-type siblings are shown to the right. (B) Section of the homozygous sal1-1-defective kernel shown in A. The cells of the supernumerary aleurone layer (al) are stained red with the lipid stain Sudan red. The highly reduced starchy endosperm (se) is shown in dark blue. (C) Micrograph showing part of a transverse section of the multilayered aleurone of the homozygous sal1-1-defective kernel shown in A.(D) Homozygous doubleal1 (sal1-1′) grains. (E) Transverse section of homozygous sal1-1′ endosperm of the grains shown in D. The two-cell-thick aleurone layer is stained red with Sudan red. (F) Micrograph showing part of the two-cell-thick aleurone of the homozygous sal1-1′ kernel shown in D. (G) Family of plants segregating for the sal1-2 allele. (H) homozygous sal1-2 plant. (I) Roots of homozygous sal1-2 plant. (J and K) Morphology of homozygous sal1-2 leaves. (L) Section of wild-type leaf. (M) Section of sal1-2 leaf.

Fig. 2.

Cloning and expression analysis of sal1.(A) DNA blot from heterozygous sal1/+ (superal) and wild-type (single aleurone layer) plants digested with BglII and probed with a Mu1-specific probe. The blot identifies a 1.6-kb BglII band cosegregating with the sal1 phenotype (arrow). (B) Relative position of Mu insertion in the two sal1 alleles. (C) DNA blot from F₂ population segregating for the sal1-2 allele probed with the sal1 DNA. (D) Northern blot analysis of sal1 expression in leaves from sal1-2/sal1-2, sal1-2/+, and +/+ plants. (Right) Northern blot analysis from wild-type kernels harvested at 10 and 20 DAP. (E) In situ hybridization analysis with sal1 antisense probe for wild-type 10 DAP peripheral endosperm. p, pericarp; al, aleurone; se, starchy endosperm. (F) Same as E with sense (control) probe. (G) In situ hybridization analysis with sal1 antisense probe on wild-type 10 DAP basal endosperm. tc, transfer cells. (H) Same as G with sense (control) probe.

Fig. 3.

SAL1 sequence and evolution. (A) Alignment of the SAL1 protein with its human homologue, CHMP1. (B) Dendrogram showing the evolutionary relationship within the vacuolar E sorting protein family. GenBank accession numbers for proteins are atSAL1 (Arabidopsis developmental protein, At1g73030); atSAL2 (Arabidopsis developmental protein, At1g17730); osSAL1 (rice, AA072640); dg1118 (Dictyostelium developmental protein, AAC67541); dmCHMP1 (Drosophila, AAL28346); CHMP1 (human, AAG01448); CHMP1.5 (human, AAG01449); CHMP2 (human, AAC00005); CHMP2.5 (human, AAD34079); CHMP4 (human, AAF29098); CHMP5 (human, AAD27743); yeast CHM1 (yeast, YKR035w); CHM2 (yeast, YKL002w); CHM5 (yeast, YDR486c); and CHM6 (yeast, YMR077c).