作为重要的农业肥料，氮肥每年的施用量超过1亿顿。提高氮肥的利用效率是整个农业生产和作物育种领域关注的重要课题。在大多数土壤中，硝态氮（NO3-）和铵态氮（NH4+）是作物吸收氮素的主要形式，植物对硝态氮吸收和转运主要通过硝酸盐转运蛋白（nitrate transporters, NRTs）和氯离子通道（chloride channel, CLC）来完成。最近，研究人员鉴定了玉米中的硝酸盐和氯化物转运蛋白NPF6的功能，为改良玉米的氮利用效率提供了新的途径。
研究人员根据玉米的序列信息分离了NPF6蛋白的两个全长cDNA Zm-NPF6.4（GRMZM2G086496_T01）和Zm-NPF6.6（GRMZM2G161459_T02）。经鉴定，Zm-NPF6.4和Zm-NPF6.4蛋白与拟南芥中的At-NPF6.3 （也叫CHL1，chlorate resistant1）同源。其亲和性转换和盐桥残基以及质子结合结构域十分保守，预示着转运蛋白的特性和功能。随后的体外试验也证明了其作为硝酸盐和氯化物转运蛋白的功能。Zm-NPF6.6对硝酸盐表现出高亲和性，对氯化物表现出低亲和性；而Zm-NPF6.4正好相反，对硝酸盐表现出低亲和性，对氯化物表现出高亲和性。对几个重要的氨基酸位点，如NPF6.6的H362、T104和NPF6.4的Y370、T106进行替换或突变，可以改变他们对硝酸盐和氯化物的亲和性。该研究系统研究了玉米NPF6蛋白的生物学功能，发现了其对硝酸盐和氯化物的转运特性。NPF6蛋白的发现和研究将为改良玉米的氮利用效率提供新的途径。
Plant Cell. 2017 Sep 8.
Maize NPF6 Proteins Are Homologs of Arabidopsis CHL1 That Are Selective for both Nitrate and Chloride.
Author
Wen Z, Tyerman S, Dechorgnat J, Ovchinnikova E, Dhugga K, Kaiser BN*.
*: Centre for Carbon Water and Food, School of Life and Environmental Sciences, University of Sydney, Australia
Abstract
Nitrate uptake by plant cells requires both high and low-affinity transport activities. Arabidopsis (Arabidopsis thaliana, At) nitrate transporter 1/peptide transporter family (NPF) 6.3 is a dual-affinity plasma membrane transport protein that has both high and low-affinity functions. At-NPF6.3 imports and senses nitrate and is regulated by phosphorylation at Thr-101 (T101). A detailed functional analysis of two maize (Zea mays, Zm) homologs of At-NPF6.3 (Zm-NPF6.6 and Zm-NPF6.4) showed that Zm-NPF6.6 was a pH dependent non-biphasic high-affinity nitrate-specific transport protein. By contrast, maize NPF6.4 was a low-affinity nitrate transporter with efflux activity. When supplied chloride, NPF6.4 switched to a high-affinity chloride selective transporter, while NPF6.6 had only a low-affinity chloride transport activity. Structural predictions identified a nitrate binding His (H362) in NPF6.6 but not in NPF6.4. Mutation of NPF6.4 Tyr-370 to His (Y370H) resulted in saturable high-affinity nitrate transport activity and nitrate selectivity. Loss of H362 in NPF6.6 (H362Y) eliminated both nitrate and chloride transport. Furthermore, alterations to Thr-104, a conserved phosphorylation site in NPF6.6, resulted in a similar high-affinity nitrate transport activity with increased Km whereas equivalent changes in NPF6.4 (T106) disrupted high-affinity chloride transport activity. NPF6 proteins exhibit different substrate specificity in plants and regulate nitrate transport affinity/selectivity using a conserved His residue.
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