The Proceedings of the National Academy of Sciences publish good science and the article reported on here is no exception.
The authors of this recent article used a recently compiled database of rice genomic variations to direct an analysis of the effect of a slight variation in the splicing of a gene transcribing a nitrate transporter in the staple crop. As nitrogen is an important influencer of cellular pH, how the differing transporters influence nitrogen uptake and cellular acidity was assessed by the researchers.
Nitrate transporter OsNRT2.3 and its alternate splicing
The researchers took the observed variation in expression of a gene named OsNRT2.3 which produced an altered protein component of a nitrate high affinity transport system. The gene is variously transcribed and spliced, the variations named OsNRT2.3a and OsNRT2.3b. The resultant protein products differ by 30 amino acids.
The starting premise of the study was that the OsNRT2.3a protein (from now on called 2.3a) is predominantly involved in transporting nitrogen from root to shoot while 2.3b on the other hand was expressed in the phloem of the shoot and only faintly expressed in the root. From this premise the researchers probed the differences between the gene products to gain an understanding of any advantages of one form over the other.
Assessing the difference between the two splices
After performing a phenotype and genotype assessment of different cultivars of rice, assessing differences in their nitrogen content and differences in expression of the two variances of this gene, they found a correlation between the ratio of expression of the two proteins and the nitrogen content of the crop.
To further explore the difference in function of the two gene products they were expressed in Xenopus oocytes (eggs of a particular species of frog which is routinely used as a vector for expressing genes for study) and subjected to different nitrate supplies, observing the changes in pH and membrane potential as the nitrogen supply changed. They found that oocytes transformed with 2.3a and its partner protein had a fluctuating membrane potential as nitrate supply changed, but this fluctuation didn’t occur in 2.3b injected oocytes.
The uptake of nitrate in oocytes transformed with 2.3b resulted in lowered cytosolic pH (found to occur naturally with nitrogen uptake) which in turn slowed the rate of nitrate uptake, a response not seen in 2.3a transformed oocytes which were not affected by the cytosolic pH. It was found that only a small lowering of pH had an affect on nitrate transport by 2.3b. Noting this effect of pH on the transporter, the protein sequences were analysed using a protein fingerprinting tool which found two pH sensing motifs in the proteins. One of the motifs was predicted to be located on different sides of the plasma membrane depending on how the gene was spliced. As described above, the pH motif was found on the cytosolic side of the plasma membrane in the 2.3b protein, but was extracellular in the 2.3a protein.
Although this predicted variation in location would explain the differing effects of pH on the two transporter, the researchers further tested this prediction by expressing the genes in rice protoplasts and using tagged antibodies to visualise the location of the histidine residues in the two proteins. This testing confirmed that the motif was on located on the inside surface of the plasma membrane in 2.3b transporters and on the outside surface in 2.3a transporter.
To test whether the predicted function of this motif matched reality, the researchers mutated the histidine residue in this motif (histidine residues noted to play an important rike in pH sensing) and found that this mutation altered the pH sensitivity of the 2.3b transporter that was previously observed.
Rice growth and nitrogen use efficiency
To test what the differences in activity meant for rice production, the two forms of the protein and the histidine mutated 2.3b protein were overexpressed and grown in field and pot experiments, monitoring growth, yield and nitrogen use efficiency compared to the wild type plants.
Rice plants overexpressing 2.3b showed improved growth, yield and nitrogen use efficiency. Testing the most overexpressed 2.3b plants by supplying only one-quarter of the normal nitrogen fertiliser amount used in commercial rice growing resulted in grain yield equal to that of wild type plants supplied with average fertiliser amounts. Further, the panicle length, number of seeds per panicle and seed setting rates were increased.
Figure from article – O lines contain overexpressed 2.3b transporters, a-O contain 2.3a overexpressed transporters, WT is wild type and H167R lines are plants with 2.3b transporters with the histidine residue mutated to arginine. Figure C shows the increases in yield and nitrogen use efficiency between the four plant types.
Nitrogen use efficiency, calculated by dividing yield by nitrogen supply, showed an efficiency increase of 26 – 47% in 2.3b overexpressed plants compared with the other two transgenic lines and the wild type.
In their discussion, the researchers confirm that both pH and membrane potential influence nitrate transport and this was seen in 2.3b. A mix of nitrogen sources between nitrate and ammonium has previously been shown to increase nitrogen uptake and it was suggested that this synergy between nitrogen sources was increased in the overexpressed 2.3b rice plants due to the internal pH sensor that wasn’t present in the other subjects.
The possible application of the research is to not only increase rice yield but also to assist growers adapt to different farming techniques that may have advantages compared to traditional water-logged growing, or to adapt to changes forced by changing climate conditions.
It is amazing that such a small change in the protein produced from a variation in the splicing of the transcribing gene results in the protein being located on alternative sides of the plasma membrane and has a marked effect on nitrate transport. But such small changes could lead to significant improvements in either or both crop yield and fertiliser use.