At present, most articles about plant single cells focus on model plants, such as Arabidopsis thaliana and rice. However, the research on tissue leaves mostly focuses on leaves and roots. The following is probably the first article I have read about tobacco single cell research, which studies corolla cells.

From June 5438 to June 20221October, New Botanist published the name of the team of South Korea's Kim Sang-kui on the Internet.

Single cell RNA sequencing of tobacco cells with pointed beak reveals a biosynthetic pathway of flora scene. " In this study, 3765 corolla cells were identified by mapping the single cell transcription map of tobacco corolla cells. By comparing the expression patterns of different genes in 3765 cells, the key genes in benzyl acetone (BA) synthesis pathway were identified: NaPAL4, Na4CL, NaPKS2/3 and NaAFR. At the same time, it was verified that BA was mainly synthesized in the epidermal layer of corolla cells, which provided a new understanding for understanding the synthesis and regulation of tobacco BA.

In this study, the author selected the samples of single cell sequencing: corolla limb and throat cup (figure A below). Then select three time points: ZT8, ZT 12, ZT 16+06. 3,756 cells were obtained by single cell sequencing, among which, according to the parameters given in the attached table: median gene per cell (about 1000) and reads map to genome (about 30%), I personally think that the single cell sequencing data quality is average.

According to the results of UMAP, it can be seen that it can be divided into 10 clusters (figure B below), and there is little difference in the distribution of clusters at three different time points (figure C below). The next step is to annotate each cluster. In this paper, the author pointed out that due to the lack of related marker genes in flowers, the author first obtained the marker genes of each cluster from scratch, and then annotated each cluster according to the marker genes. Among them, the main marker genes are: cluster 7 (pollen: pectinesterase gene, extension family and pollen allergen), cluster 8 (phloem/xylem: sweet112, Sudan 2; 1), cluster 0/3/4/5/9 (epidermal cells: ketoacyl-coa synthetase, ECERIFERUM4, wax ester synthetase/diacylglycerol acyltransferase 1, lipid transfer protein, ethylene glycol -3- phosphate acyltransferase 6, keratin synthetase-like) (Figure D), cluster 6 (Figure D)

Next, the author focuses on the synthesis of ba. In corolla, the pathway of BA synthesis starts from NaPAL4 (figure A below). Then 4CL/CNL enzyme is needed next. The next step is completed by NaPKS (Tobacco has four members 1/2/3/4, but it is not confirmed which member). Except for PAL4, PKS doesn't know which member it is, neither 4CL nor AER is clear and has not been confirmed. Therefore, based on known PAL4, the authors hypothesized that other genes in the synthetic pathway should have strong correlation with PAL4 at the single cell level, or that the expression of patter is consistent (this is the same as the study of genes and gene regulation).

Therefore, the author calculated the correlation between all genes identified by single cell sequencing and PAL4 (Figure B below), and made synchronous bidirectional verification with published RNA-seq data. From the results, the results of single cell are consistent with those of RNA-seq, such as 4CL 1 gene, PKS2/3, AER 1. However, there are also some inconsistencies. For example, from the perspective of correlation distribution, single cells are more concentrated and more in line with normal distribution (figure C below). And IFR3 is very different.

Then, the author began to verify the role of 4CL 1, PKS2/3 and AER 1 genes in BA synthesis.

The figure below shows the result of 4CL 1 knockout. From the point of view of VIGS knockout, the expression of 4CL 1 in the top and inside of corolla decreased significantly (below a/b), and the content of BA also decreased significantly (below a/b). The metabolites are cinnamic acid and coumaric acid, not caffeic acid and ferulic acid. In addition, the author also checked that other 4CL members were not affected in this knock-out material of 4CL 1.

Then, the author tries to verify the function of PKS2/3. From the knock-out material of PKS2/3, it can be seen that the content of BA is obviously reduced both at the top and inside (a/b below). However, due to the high similarity of PKS family, PKS 1/4 also decreased in knockout materials. At that time, when the author added PKS2 and PKS3 to the product of 4CL 1, it was found that BA was identified only in the reactant added with PKS2, but not in PKS3 (figure C below). It shows that the gene PKS2 plays a key role in this process, and the correlation of PKS2 is far stronger than that of PKS3.

The function of AER 1 in BA synthesis was also tested. Similarly, in the knockout material of AER 1, the BA content in the top and inside of the corolla decreased obviously (below a/b). In addition, a new benzylidene acetone compound (figure C below) was found in the gene knockout plants of AER 1, which is a non-reducing form of BA. However, this new compound does not exist at the top of wild-type corolla (figure C below).

After proving the role of these four genes in BA synthesis (in fact, it seems that the discovery process of these four genes does not need single cell data, and ordinary multi-tissue RNA-seq can also be realized, after all, the results of RNA-seq of these four genes are also included), the author began to explore the distribution law of these four genes in different cells by using single cell data. Then it was found that among 3756 cells, 1097 cells contained these four genes, and these 1097 cells were basically concentrated in 0/3/4 cluster (figure A below). From the expression patterns of four genes in different cells, it can be clearly found that they are mainly enriched in 0/3/4 cluster (b/c below). It is known that these three clusters are epidermal cells, so it is reasonable to assume and guess that BA may be synthesized in epidermal cells of corolla.

GFP localization information also showed that these genes were mainly expressed in epidermal cells (below).

Because the synthesis of BA in corolla is regulated by biological clock, the author looked at the regulation relationship between these four key functional genes and biological clock rhythm. From the expression pattern, the expression of these four functional genes changed rhythmically with time (Figure A below), which is similar to the known rhythm genes (Figure A below), as is the change of BA content (Figure A below). Because single cell sampling needs three time points, including day and night points. Therefore, the author also looked at the changes of these four genes in single cell data, and it can be clearly seen that there is a peak at 12h during the day (figure B below). Then, the differences of BA and four key synthetic genes in corolla branches and corolla tubes were determined. As can be seen from the content of the combination, Ba is mainly in limbs. From the expression point of view, although PAL and 4CL are also expressed in the test tube, PKS2 and AERA are almost not expressed in the test tube, which proves the synthesis site of BA again (figure C below).