An invertase inhibitor from maize localizes to the embryo surrounding region during early kernel development

Abstract

Invertase activity is thought to play a regulatory role during early kernel development by converting sucrose originating from source leaves into hexoses to support cell division in the endosperm and embryo. Invertases are regulated at the posttranslational level by small protein inhibitors, INVINHs. We found that in maize (Zea mays), an invertase inhibitor homolog (ZM-INVINH1) is expressed early in kernel development, between 4 and 7 d after pollination. Invertase activity is reduced in vitro in the presence of recombinant ZM-INVINH1, and inhibition is attenuated by pre-incubation with sucrose. The presence of a putative signal peptide, fractionation experiments, and ZM-INVINH1::green fluorescent protein fusion experiments indicate that the protein is exported to the apoplast. Moreover, association of ZM-INVINH1 with the glycoprotein fraction by concanavalin A chromatogaphy suggests that ZM-INVINH1 interacts with an apoplastic invertase during early kernel development. ZM-INVINH1 was localized to the embryo surrounding region by in situ analysis, suggesting that this region forms a boundary, compartmentalizing apoplast invertase activity to allow different embryo and endosperm developmental rates.

Figure 1.

Nucleotide and deduced amino acid sequence of the ZM-INVINH1 gene, including 587 bp upstream of the ATG. The deduced amino acid sequence is presented above the DNA sequence. Putative TATA sites are underlined, and the site of the longest cDNA clone is indicated by a double underline.

Figure 2.

Comparison of ZM-INVINH1 with other invertase inhibitor-like proteins. Alignment of the deduced amino acid sequence of INVINH-like proteins from maize (ZM-INVINH1, ZM-INVINH2, and ZM-INVINH3), wheat (TA-INVINH1 and TA-INVINH2), rice (OS-INVINH1, OS-INVINH2, and OS-INVINH3), as well as previously published sequences from tobacco (NT-INVINH1 and NT-INVINHH), tomato (Lycopersicon esculentum; LE-INVINH1), and Arabidopsis (AT-INVINHH). Conserved Cys residues are indicated by asterisks.

Figure 3.

Recombinant ZM-INVINH1 inhibits invertase activity in vitro. ZM-INVINH1 produced in Escherichia coli was pre-incubated with an enzyme extraction from maize kernels harvested at 10 DAP. A, Invertase activity measured after pre-incubation with 20 to 200 pmol of recombinant ZM-INVINH1. B, Effect of Suc on ZM-INVINH1 inhibition of invertase activity. Pre-incubation of 10-DAP kernel enzyme extract with 50 pmol of ZM-INVINH1 and 1 or 5 mm Suc. The data are presented as a mean ± sd of three reactions.

Figure 4.

Fractionation of ZM-INVINH1. A, Proteins isolated from 5-DAP (odd lanes) and 10-DAP (even lanes) kernels were fractionated into soluble (lanes 1 and 2), high-salt wash (lanes 3 and 4), and insoluble fractions (lanes 5 and 6) and subjected to western-blot analysis with antibody raised against ZM-INVINH1. Fifty micrograms of total soluble protein and 20 μg of high-salt and insoluble protein were separated by SDS-PAGE, and western-blot analysis was performed with ZM-INVINH1 antibody. B, ZM-INVINH1 associates with the glycoprotein fraction in maize kernels. Glycoproteins were isolated from 5- and 10-DAP kernels by ConA chromatography, and ZM-INVINH1 was detected by western-blot analysis. Lanes 1 and 2 correspond to ConA chromatography flow through and eluate, respectively. A large arrow represents ZM-INVINH1, and an asterisk indicates a high M_r cross-reacting protein.

Figure 5.

ZM-INVINH1 is targeted to the apoplast. Particle bombardment into onion epidermal cells was performed with GFP alone (A and B) or fused to ZM-INVINH1 at the C terminus (E and F) under the control of the maize ubiquitin promoter. Cytoplasmic streams and the nucleus evident in GFP bombardments are indicated by cs and n, respectively.

Figure 6.

Expression analysis of ZM-INVINH1 by RT-PCR. Top, Total RNA isolated from mature leaf (L), root (R), and tassel (T), as well as from whole kernels harvested 4, 7, 10, and 15 DAP was subjected to RT-PCR to amplify the ZM-INVINH1 transcript. A M_r marker (M) is included. Bottom, A control reaction using primers designed to amplify maize tubulin was also performed.

Figure 7.

In situ analysis of ZM-INVINH1 in 5-DAP kernels. Embedded and longitudinal kernel sections were hybridized with an antisense (A-C) or sense transcript (D-F) generated from ZM-INVINH1. ped, pedicel; n, nucellus; p, pericarp; en, endosperm; em, embryo.