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zmsbt1 and zmsbt2, two new subtilisin-like serine proteases genes expressed in early maize kernel development

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Abstract

Main conclusion

Two subtilisin-like proteases show highly specific and complementary expression patterns in developing grains. These genes label the complete surface of the filial–maternal interface, suggesting a role in filial epithelial differentiation.

The cereal endosperm is the most important source of nutrition and raw materials for mankind, as well as the storage compartment enabling initial growth of the germinating plantlets. The development of the different cell types in this tissue is regulated environmentally, genetically and epigenetically, resulting in the formation of top–bottom, adaxial–abaxial and surface–central axes. However, the mechanisms governing the interactions among the different inputs are mostly unknown. We have screened a kernel cDNA library for tissue-specific transcripts as initial step to identify genes relevant in cell differentiation. We report here on the isolation of two maize subtilisin-related genes that show grain-specific, surficial expression. zmsbt1 (Zea mays Subtilisin1) is expressed at the developing aleurone in a time-regulated manner, while zmsbt2 concentrates at the pedicel in front of the endosperm basal transfer layer. We have shown that their presence, early in the maize caryopsis development, is dependent on proper initial tissue determination, and have isolated their promoters to produce transgenic reporter lines that assist in the study of their regulation.

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Abbreviations

BETL:

Basal endosperm transfer cell layer

DAP:

Days after pollination

GUS:

β-Glucuronidase

ISH:

In situ hybridization

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Acknowledgements

This work was supported by a grant from the Spanish Ministerio de Ciencia e Innovacion (BIO2012-39822) and a contract of the European Commission (QLK3-CT-2000-00302) to Gregorio Hueros and internal funds from Biogemma SAS. We thank Y. Sanz for excellent technical support.

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Correspondence to Gregorio Hueros.

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425_2016_2615_MOESM1_ESM.tif

Suppl. Fig. S1 Expression analyses of zmsbt1, zmsbt2 and GRMZM2G363552 along maize kernel development, based on published RNA-Seq data (Chen et al. 2014). The Y axis shows expression level as RPKM (Reads Per Kilobase per Million mapped reads) values. The genes were not detected in any plant tissue outside the kernel. In the kernel, no signal was also found in the embryo (Em) at any developmental stage tested (from 10 to 32 DAP). zmsbt1 is expressed in the endosperm (En) between 6 and 10 DAP and, consequently, in the complete grains (S) at the same developmental stages. zmsbt2 and GRMZM2G363552 were exclusively expressed in the complete caryopsis, from 3 to 10 DAP (TIFF 3551 kb)

425_2016_2615_MOESM2_ESM.tif

Suppl. Fig. S2 Expression analyses of zmsbt1, zmsbt2 and GRMZM2G363552 in different compartments within the maize kernel, based on published RNA-Seq data (Zhan et al. 2015). The Y axis shows expression level as RPKM (Reads Per Kilobase per Million mapped reads) values. The scale has been broken to facilitate data visualization. Al, aleurone; BETL, basal endosperm transfer cell layer; CSE, central starchy endosperm; CZ, conductive zone; EMB, embryo; ESR, embryo surrounding region; Nu, nucellus; PC, placenta-chalaza; PE, pericarp; PED, pedicel. zmsbt1 is almost exclusively found in the aleurone. zmsbt2 and GRMZM2G363552 were detected in the placenta-chalaza and, at a minor extend, in the pedicel (TIFF 3044 kb)

425_2016_2615_MOESM3_ESM.tif

Suppl. Fig. S3 zmsbt1 expression is linked to a differentiating aleurone. Antisense probes were hybridized to wild type (panels a, c for zmsbt1; panels e, g for BETL-9like) and dek1 (panels b, d for zmsbt1; panels f, h for BETL-9like) grain sections of 7 (a, b, e, f) and 15 DAP (c, d, g, h). Only the upper part of the endosperms is shown in all images. Note that the reduced zmsbt1 signal at 15 DAP in WT material is due to low expression at this point, as tissue identity is confirmed by the strong BETL-9like detection. Al, aleurone; En, endosperm. Bars = 500 μm (a, b, c, f, g, h), 250 μm (d, e) (TIFF 5809 kb)

425_2016_2615_MOESM4_ESM.tif

Suppl. Fig. S4 zmsbt1 does not accumulate in cr4 aleurone. A zmsbt1 antisense probe was hybridized to wild type (a, b) and cr4 (c, d) kernel sections of 5 (a, c) and 11 DAP (b, d). At 5 DAP both genotypes present signal at the adgerminal side of the aleurone layer, although the signal appears to extend to additional cell layers in cr4 endosperms. At 11 DAP, however, both genotypes differ markedly, with the mutant displaying the signal in a discontinuous manner while wild type kernels display a strong expression along the whole aleurone layer. The squared areas are shown at higher magnification in the lower right corner of each panel. Arrows indicate the position of the aleurone layer. En, endosperm. Bars = 100 µm (a and c), 1000 µm (b and d) and 50 µm in the insets (TIFF 4728 kb)

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López, M., Gómez, E., Faye, C. et al. zmsbt1 and zmsbt2, two new subtilisin-like serine proteases genes expressed in early maize kernel development. Planta 245, 409–424 (2017). https://doi.org/10.1007/s00425-016-2615-2

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