shrunken4 is a mutant allele of ZmYSL2 that affects aleurone development and starch synthesis in maize

Abstract

Minerals are stored in the aleurone layer and embryo during maize seed development, but how they affect endosperm development and activity is unclear. Here, we cloned the gene underlying the classic maize kernel mutant shrunken4 (sh4) and found that it encodes the YELLOW STRIPE-LIKE oligopeptide metal transporter ZmYSL2. sh4 kernels had a shrunken phenotype with developmental defects in the aleurone layer and starchy endosperm cells. ZmYSL2 showed iron and zinc transporter activity in Xenopus laevis oocytes. Analysis using a specific antibody indicated that ZmYSL2 predominately accumulated in the aleurone and sub-aleurone layers in endosperm and the scutellum in embryos. Specific iron deposition was observed in the aleurone layer in wild-type kernels. In sh4, however, the outermost monolayer of endosperm cells failed to accumulate iron and lost aleurone cell characteristics, indicating that proper functioning of ZmYSL2 and iron accumulation are essential for aleurone cell development. Transcriptome analysis of sh4 endosperm revealed that loss of ZmYSL2 function affects the expression of genes involved in starch synthesis and degradation processes, which is consistent with the delayed development and premature degradation of starch grains in sh4 kernels. Therefore, ZmYSL2 is critical for aleurone cell development and starchy endosperm cell activity during maize seed development.

Keywords: aleurone; iron transporter; maize; seed development; starch synthesis.

Figure 1

The maize kernel mutant shrunken4 (sh4) exhibits a starch-deficient phenotype. (A) Mature F₂ ear from a cross between sh4 and W22. Arrows indicate sh4 kernels. Bar = 1 cm. (B) Phenotypic observation and longitudinal sections of wild-type (WT) and sh4 mature kernels from a segregated F₂ ear. The mature kernels were viewed under natural light (left), under transmission light (middle), and as longitudinal sections (right). En, endosperm; Em, embryo. Bars = 1 mm. (C) Germination test of WT and sh4 mature kernels (7 DAG). Bar = 1 cm. (D) Developing F₂ ear of sh4 × W22. Arrows indicate sh4 kernels. Bar = 1 cm. (E) Longitudinal sections of developing wild-type and sh4 kernels at 15 DAP. Bar = 1 mm. (F,G) Comparison of the 100-kernel weight of randomly selected mature WT and sh4 kernels (F) and the starch contents of mature WT and sh4 endosperm from individual kernels (G). Values are means ± SE; n = 3 (***, P < 0.001; Student’s t-test).

Figure 2

Positional cloning and genetic confirmation of Sh4. (A) The sh4 mutant was crossed to the B73 inbred line, and the progeny were selfed to obtain F₂ ears. A total of 6000 sh4 homozygous kernels from the F₂ population were analyzed. The numbers below each molecular marker indicate the ratio of recombinant kernels in the tested population. The sh4 locus was narrowed down to a ∼43-Kb interval on chromosome 5 containing two candidate genes: Zm00001d017426 and Zm00001d017427. (B) Schematic of the structure and mutation site of the Zm00001d017427 gene. Lines and black boxes indicate introns and exons, respectively. The open boxes indicate the 3’ and 5’ untranslated regions. (C–E) Functional complementation of sh4 via transformation. (C) Characteristic kernels with wild type (1–10) and shrunken and opaque mutant (11–20) phenotypes from F₂ ears produced from a cross between a Zm00001d017427-ORF-expressing transgenic plants (T₀) and an sh4 heterozygous plant. The kernels were observed under natural light (1–5 and 11–15) and transmission light (6–10 and 16–20). (D) Detection of homozygous sh4 kernels using the molecular marker SSR873-1. (E) Identification of transgenic kernels using primers for the Bar gene. −, water control; +, Zm00001d017427 transgene construct. (F,H) CRISPR-Cas9-based mutation of Zm00001d017427 and allelism test with sh4. (F) The targeted sequence in the second exon of Zm00001d017427 using CRISPR/Cas9. gRNA, guide RNA; PAM, protospacer-adjacent motif. (G) Allelism test ear produced from a cross between sh4-cas9-1/+ and sh4/+. Arrows indicate mutant kernels. Bar = 1 cm. (H) Randomly selected kernels from (G). Bar = 1 cm.

Figure 3

Transport activity of Sh4/ZmYSL2. (A) Phylogenetic analysis of Sh4/ZmYSL2 and its homologs. The neighbor-joining tree was constructed with MEGA 7.0. The numbers at the branches indicate the percentage of 1000 bootstraps. Scale bar indicates the average number of amino acid substitutions per site. (B) Comparison of iron concentration in fet3fet4 yeast cells expressing empty vector, ZmYSL2, or YS1. Values are means ± SE; n = 3 (**, P < 0.01; ***, P < 0.001; Student’s t-test). (C) Results of voltage-clamp experiments analyzing ZmYSL2 in X. laevis oocytes. The oocytes were sequentially examined in three different buffers [ND96 buffer as a control, Zn²⁺-NA (100 μM) in ND96 buffer, and Fe²⁺-NA (100 μM) in ND96 buffer] after 15 minutes of washing with ND96 buffer. The current-voltage curves were recorded in oocytes expressing ZmYSL2 in control, Zn²⁺-NA, and Fe²⁺-NA buffer.

Figure 4

Sh4/ZmYSL2 is concentrated in the aleurone layer. (A,B) ZmYSL2 protein content in developing kernels over time (A) and in different tissues (B). α-Actin was used as an internal standard. The developing kernels in (A) were collected at different stages and labeled according to DAP. Pericarp (PE), embryo (Em), and endosperm (En) samples in (B) were collected at 15 DAP. (C) Immunoblot comparing the ZmYSL2 protein content in WT, sh4, and sh4-cas9-1 endosperm. α-Actin was used as an internal standard. (D) Immunostaining with anti-ZmYSL2 antibody in a longitudinal section of a 15 DAP developing WT kernel. Bar = 1 mm. The inset shows an enlarged image of the boxed region; bar = 100 μm. SC, scutellum; LP, leaf primordia; SAM, shoot apical meristem; RAM, root apical meristem; AL, aleurone layer; SAl, sub-aleurone layer.

Figure 5

The sh4 mutation alters aleurone cell fate in maize endosperm. (A) Comparison of iron concentrations in mature WT and sh4 whole kernels, endosperm, and embryos. Values are means ± SE; n = 3 (***, P < 0.001, Student’s t-test). (B) Iron localization in the aleurone layer of 15 DAP WT and sh4 kernels by Perl’s staining. AL, aleurone layer; En, endosperm. Bars = 100 μm. (C) Cell morphology in the aleurone layer in wild type and sh4 endosperm at 15 DAP. Bars = 100 μm. (D) TEM showing the aleurone layer and three endosperm cell layers near the aleurone layer. Bars = 10 μm. (E) Cell morphology of the aleurone layer in wild type and sh4 endosperm under TEM. AG, aleurone granule; SG, starch granule. Bars = 5 µm.

Figure 6

Loss of Sh4/ZmYSL2 function alters the expression of genes involved in storage protein and starch synthesis. (A) The most significant GO terms of the DEGs based on RNA-seq analysis of 15 DAP wild type and sh4 endosperm. (B) Heatmap showing the changes in the expression levels of storage-protein-related genes in WT and sh4 endosperm. (C) Immunoblot comparing the storage protein contents in WT and sh4 endosperm. α-Tubulin was used as an internal standard. (D) Heatmap showing the changes in expression levels of starch-synthesis-related genes in WT and sh4 endosperm. The expression levels of these genes are listed in Data S3. UDPG, Uridine 5’ diphosphate-glucose; G1P, glucose 1-phosphate; G6P, glucose 6-phosphate; ADPG, Adenosine 5’ diphosphate-glucose; Sus, sucrose synthase; Ugp, UDP-glucose pyrophosphorylases; Pgm, phosphoglucomutase; Gpt, glucose-6-phosphate/phosphate translocator; AGPL, ADP-glucose pyrophosphorylase large subunit; AGPS, ADP-glucose pyrophosphorylase small subunit; SS, starch synthases; SBE, starch branching enzyme; DBE, starch debranching enzyme; Ae1, amylose extender1; Su1, Sugary1; ISA, Isoamylase; Zpu1, Z. mays pullulanase-type starch debranching enzyme1; Wx1, Waxy1; Pho, Phosphorylase.

Figure 7

Loss of Sh4/ZmYSL2 function leads to delayed formation and premature degradation of starch grains in maize endosperm. (A) Comparison of glucose, fructose, and sucrose contents of developing WT and sh4 kernels. Values are means ± SE; n = 3 (***, P < 0.001, Student’s t-test). (B) Iodine stained longitudinal paraffin sections of developing kernels. Bar = 1 mm. (C) Magnified images of starchy endosperm cells at 15 DAP. Bars = 100 µm. (D) Images of the fourth starchy endosperm cell layer from the aleurone layer under a transmission electron microscope (TEM). Bars = 10 μm. (E) Scanning electron microscopy of WT and sh4 developing endosperm. En, endosperm; PB, protein body; SG, starch granule. Bars = 5 µm.

Figure 8

A model of the role of Sh4/ZmYSL2 in maize kernel development. AL, aleurone layer; AG, aleurone granule; SG, starch granule; PB, protein body.