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Maize (Zea mays) requires substantial amounts of nitrogen, posing a challenge for its cultivation. Recent work discovered that some ancient Mexican maize landraces harbored diazotrophic bacteria in mucilage secreted by their aerial roots. To see if this trait is retained in modern maize, we conducted a field study of aerial root mucilage (ARM) in 258 inbred lines. We observed that ARM secretion is common in modern maize, but the amount significantly varies, and only a few lines have retained the nitrogen-fixing traits found in ancient landraces. The mucilage of the high-ARM inbred line HN5-724 had high nitrogen-fixing enzyme activity and abundant diazotrophic bacteria. Our genome-wide association study identified 17 candidate genes associated with ARM across three environments. Knockouts of one candidate gene, the subtilase family gene ZmSBT3, confirmed that it negatively regulates ARM secretion. Notably, the ZmSBT3 knockout lines had increased biomass and total nitrogen accumulation under nitrogen-free culture conditions. High ARM was associated with three ZmSBT3 haplotypes that were gradually lost during maize domestication, being retained in only a few modern inbred lines such as HN5-724. In summary, our results identify ZmSBT3 as a potential tool for enhancing ARM, and thus nitrogen fixation, in maize., (© 2023 The Authors. Journal of Integrative Plant Biology published by John Wiley & Sons Australia, Ltd on behalf of Institute of Botany, Chinese Academy of Sciences.)

Background: The mesocotyl connects the coleoptilar node and the basal part of the seminal root of maize (Zea mays) seedling. The mesocotyl pushes the shoot of the seedling out of the soil during seed germination; thus, its growth is highly related to deep-sowing tolerance. Although many studies on the maize mesocotyl have been carried out at physiological and molecular levels, the proteomic changes associated with cellular and physiological activities during mesocotyl growth are still unknown., Results: In the present study, the maize hybrid Zhengdan 958 was used to study mesocotyl growth and accompanying protein changes. The dark-grown etiolated mesocotyls exhibited a slow-fast-slow feature, with significant changes in the levels of indole-3-acetic acid (IAA) and cellulose and the activity of peroxidase (POD). In particular, POD activity increased with mesocotyl growth, showing higher activity at the mature (lower) end of the mesocotyl. For the proteomic analysis, soluble proteins were extracted from etiolated mesocotyls dark-grown for 48 h, 84 h, and 132 h, corresponding to the initial, rapid, and slow growth periods, respectively, and subjected to separation by two-dimensional gel electrophoresis (2-DE). As a result, 88 differentially abundant proteins (DAPs) were identified using MALDI-TOF-TOF analysis. At 48 h, most DAPs were stress proteins, heat shock proteins and storage proteins; at 84 h, oxidation/reduction proteins, carbohydrate biogenesis-related proteins and cytoskeleton-related proteins were highly accumulated; at 132 h, the most striking DAPs were those involved in the synthesis and modification of the cell wall and the biogenesis of carbohydrates. Gene ontology (GO) analysis showed that changes in the abundance and proportion of DAPs were consistent with cellular and physiological activities and biological processes during mesocotyl growth. The accumulation of nine DAPs of interest was verified by immunoblotting and RT-qPCR., Conclusions: The present study revealed that the protein patterns in 2-D gels differed greatly with mesocotyl growth. At different growth periods, a specific set of DAPs participate in various biological processes and underlie the cellular and physiological activities of the mesocotyl. These results contributed to the understanding of mesocotyl growth and the cultivation of maize lines with deep-sowing tolerance.

Protein extracts obtained from cells or tissues often require removal of interfering substances for the preparation of high-quality protein samples in proteomic analysis. A number of protein extraction methods have been applied to various biological samples. TCA/acetone precipitation and phenol extraction, a common method of protein extraction, is thought to minimize protein degradation and activity of proteases as well as reduce contaminants like salts and polyphenols. However, the TCA/acetone precipitation method relies on the complete pulverization and repeated rinsing of tissue powder to remove the interfering substances, which is laborious and time-consuming. In addition, by prolonged incubation in TCA/acetone, the precipitated proteins are more difficult to re-dissolve. We have described a modified method of TCA/acetone precipitation of plant proteins for proteomic analysis. Proteins of cells or tissues were extracted using SDS-containing buffer, precipitated with equal volume of 20% TCA/acetone, and washed with acetone. Compared to classical TCA/acetone precipitation and simple acetone precipitation, this protocol generates comparable yields, spot numbers, and proteome profiling, but takes less time (ca. 45 min), thus avoiding excess protein modification and degradation after extended-period incubation in TCA/acetone or acetone. The modified TCA/acetone precipitation method is simple, fast, and suitable for proteomic analysis of various plant tissues in proteomic analysis., Competing Interests: The authors have declared that no competing interests exist.