SiAAP9 exhibits two alternative splicing events.
Previous study (Yang et al. 2021) has shown that SiAAP9 may highly expressed in immature spikelets and seeds during the filling stage. We supposed that SiAAP9 may play an important role in regulating seed development. At first, SiAAP9 was cloned using cDNA templates of Jingu 21 (Fig. 1A). Surprisingly, the bands of the colony PCR that linked SiAAP9 coding sequence to the CV16 plasmid cloning vector were not on the same horizontal line (Fig. 1B). Through sequence alignment, it was found that SiAAP9 lacked a sequence on Exon4 (Fig. 1C, supplementary Fig. S1A). Therefore, we labeled the long sequence as SiAAP9L and the short sequence as SiAAP9S. Additionally, AAP transporters have previously been reported as plasma membrane transporters in Arabidopsis and rice (Ji et al. 2020; Santiago and Tegeder 2016), we predicted SiAAP9 transmembrane domain and protein tertiary structure. Notably, we observed that the protein encoded by the long transcript exhibited an extra extensive loop structure spanning amino acids 200 to 250 in contrast to the protein encoded by the short transcript (supplementary Fig. S1C, D, E, F).
The expression patterns Analysis of SiAAP9.
To detect the complete, long and short transcripts of SiAAP9, specific primers were designed (Fig. 2A). RT-qPCR analysis of different tissues of Jingu 21 showed that the expression levels of the two transcripts were higher in shoot and lower in leaves, flowers and grains (Fig. 2B-D). In order to investigate whether SiAAP9 is induced by short-term NO3- treatment, we performed RT-qPCR analysis and found that after 1 hour of NO3- addition, the expression level of SiAAP9 two transcripts showed a significant increase, while there was no significant change after 1 hour of NO3- reduction (Fig. 2E-J). This suggests that SiAAP9 is induced by short-term NO3- exposure. Additionally, transgenic Arabidopsis plants were used to observe the expression of SiAAP9. The results of GUS staining revealed that SiAAP9 is expressed in all tissues throughout developmental stages, including seedlings (supplementary Fig. S2A), leaves (supplementary Fig. S2B, D), stomata (supplementary Fig. S2E), flower organs (supplementary Fig. S2F), pistil (supplementary Fig. S2G), root (supplementary Fig. S2H), stamen (supplementary Fig. S2I) and developing seeds (supplementary Fig.S2J). However, SiAAP9 showed low expression in the root tip (supplementary Fig. S2C).
SiAAP9 inhibits growth and development in transgenic Arabidopsis.
To further investigate the function of SiAAP9, we generated transgenic plants by overexpressing SiAAP9 in Arabidopsis using the 35S promoter. Long and short transcripts each selected two independent T3 homozygous overexpressing lines, namely LOX-1#, LOX-2#, SOX-1# and SOX-2#. These lines were identified through RT-qPCR and immunoblotting analysis (supplementary Fig. S3). For subsequent experiments, we specifically selected two lines with significantly different expression levels in SiAAP9L. Throughout the entire growth and development period of the transgenic plants, LOX-1# showed no significant difference compared to the WT in agronomic traits, the other lines exhibited a significant decrease in growth compared to the WT. The primary root length at 7 days of LOX-1# increased by 4.3%, while the other three lines decreased by 5.0%, 5.9% and 3.4% respectively compared to WT. There was no significant difference in the rosette leaves area at 3 weeks between LOX-1# and WT, while the other three lines also significantly decreased by 56%, 16.8% and 20.1%, respectively. And plant height at 4-8 weeks displayed obviously dwarfism (Fig. 3). The size and weight of grains have a close relationship with crop yield and quality (Hori and Sun 2022; Li et al. 2022). Additionally, the seed size and 1000-seed weight were also suppressed in SiAAP9 transgenic plants compared to those in Col-0, in which LOX-2# has a more significant reduction compared to other lines (Fig. 4A-D), indicating that SiAAP9 negatively regulates the size and weight of seeds. To further investigate whether overexpression of SiAAP9 affects the content of amino acids in seeds, we measured the content of 20 amino acids in seeds and found that the amino acid content in overexpressed SiAAP9 seeds was significantly higher than that in WT, except for Arg, Asn and Asp. Notably, LOX-2# and SOX-2# have significantly higher content of most amino acids than WT (supplementary Fig. S4). These results indicate that SiAAP9 has an inhibitory effect on the development of vegetative organs, but increases the amino acid content in the seeds, may increase the nutritional quality potentially.
The effect of SiAAP9 on primary root growth is determined by its expression level, regardless of nitrogen availability.
Previous studies have shown that under nitrogen sufficient conditions, there is no significant difference in the primary root length between overexpressing GmAAP6a and WT. However, under nitrogen free conditions, the primary root length of overexpressing GmAAP6a is significantly longer than WT (Liu et al. 2020). Hence, we aim to explore the impact of varying nitrogen levels on the biological function of SiAAP9.
In this study, we investigated the response of SiAAP9 to NO3- and NO3- free. When exposed to NO3- free stress, SiAAP9 overexpression plants have smaller aerial parts and fewer and shorter lateral roots in the primary root. Furthermore, after being cultured on a nitrogen rich medium for 4 days and then transferred to NO3- free medium for 8 days, we observed that the primary root length, root variation length and fresh weight of LOX-1# were significantly increased than WT, while LOX-2# and SOX-1# lines were significantly reduced than WT. However, SOX-2# showed no significant changes (Fig. 5). Interestingly, the seedlings transferred from 0 mM NO3- medium for 4 days to 7 mM NO3- medium for 8 days showed the same significant change trend (supplementary Fig. S5). These results indicate that low expression levels of SiAAP9 can promote the growth and development of primary roots, while high expression levels of SiAAP9 can inhibit the growth of primary roots. And we concluded that the growth and development of primary roots are mainly controlled by SiAAP9 expression levels, rather than long-term NO3- regulation.
SiAAP9 is insensitive to excessive Glu and His.
In order to clarify the amino acid substrates that SiAAP9 may transport, we conducted an investigation on the sensitivity of SiAAP9 transgenic Arabidopsis plants to different types of amino acids. By creating various amino acid concentration gradients, we observed phenotypic differences when exposed to 10 mM Glu and 5 mM His (Fig. 6A-B), the results showed that the overexpressing SiAAP9 lines had larger aboveground parts and longer roots compared to WT. While overexpressing SiAAP9 lines showing no sensitivity to the other 17 amino acids (supplementary Fig. S6, S7). To eliminate the interference of inorganic nitrogen, we investigated the sensitivity of SiAAP9 to amino acids in medium with amino acids as the sole nitrogen source, we discovered that both the wild type and the overexpression plants wilted on a medium with Glu as the sole nitrogen source (Fig. 6C, E, F). Conversely, on the medium with His as the sole nitrogen source, the root length and fresh weight of the overexpression transgenic lines significantly increased compared to WT (Fig. 6D, G, H). These results indicate that SiAAP9 overexpressing plants become more sensitive to excessive Glu after lacking inorganic nitrogen, while remaining insensitive to excessive His.
The two proteins encoded by SiAAP9L and SiAAP9S have different subcellular localization.
To detect the subcellular localization of SiAAP9S and SiAAP9L and the effect of nitrogen on subcellular localization, 4-day-old seedlings cultured on 7 mM NO3- or 0 mM NO3- medium were used to figure it out. We found that LOX-1# is only localized on the plasma membrane, other organelles are basically undetectable and can only be observed under specific conditions (Fig. 7A). Compared to LOX-1#, SiAAP9 may localized on organelles in other lines (Fig. 7B-D). Furthermore, by analyzing the fluorescence intensity of 40 cells, we observed that cells exposed to 0 mM NO3- exhibited stronger fluorescence signals compared to those exposed to 7 mM NO3-. To further explore the specific localization of SiAAP9L and SiAAP9S, we utilized FM4-64 dye to locate on different organelles. The staining results indicate that LOX-1# has a high degree of plasma membrane localization, while other lines also have partial colocalization on TGN and PVC in addition to plasma membrane localization. Based on this observation, we hypothesize that a higher expression level of SiAAP9 may disrupt the protein sorting process. It is known that BFA sensitive transport pathways consist of the efflux pathway from the Golgi to the plasma membrane and the degradation pathway from the Golgi to the vacuole (Pimpl et al. 2003). Surprisingly, after BFA treatment, we found that SiAAP9L exhibits significant colocalization with BFA compartment, while SiAAP9S does not. This suggests that
sequence deletion weakens the sensitivity of SiAAP9 to BFA. We did not observe any significant effect of WM treatment that can cause the fusion of vacuolar precursors to become larger (Wang et al. 2009) (supplementary Fig. S8A-D). On the other hand, we speculate that SiAAP9 protein is detained on ER, and ER staining results indicate that both SiAAP9L and SiAAP9S are localized on ER (supplementary Fig. S8E). Overall, the high expression of SiAAP9 alters the subcellular localization of its encoded protein, with differences in localization between long and short proteins.
SiAAP9 may promote Glu uptake in Jingu 21 protoplast.
In order to further validate the transport of Glu by SiAAP9, we conducted a protoplast amino acid uptake assay. Empty vector, OE-SiAAP9L, and OE-SiAAP9S transformed into protoplasts of Jingu 21 were cultured under dark conditions for 4 hours using FITC labeled Glu. The results showed that the fluorescence intensity of protoplasts transformed with OE-SiAAP9 was significantly higher than that of protoplasts transformed empty vector (Fig. 8), indicating that SiAAP9 can transport more Glu into foxtail millet cells.