The Arabidopsis thaliana wild type (WT) used in this study was the Columbia-0 (Col-0) ecotype. It was purchased from ABRC. The two overexpression lines OE4 and OE5 were the same as the 35S:AtTPPI transgenic lines mentioned previously (Lin et al., 2020). The seeds were all in our library from Jiangsu Normal University. We ensure that our experimental research on plants; including the collection of plant material, comply with relevant institutional, national and international guidelines and legislation.

The seeds of these lines were surface sterilized and sown vertically on Murashige and Skoog (MS) media (supplemented with 1% sucrose and 6 g L^–1 agar) and stored at 4°C for three days. Then, all the plates were transferred to a plant tissue culture chamber under a 16 h light/8 h dark photoperiod, a 22°C temperature, a 120 μmol quanta m⁻² s⁻¹ light intensity and 50% relative humidity.

For the vertical MS media culture, the plates were all placed in a plant tissue culture chamber, and after 14 days of growth, images were taken. Afterward, the roots were removed and measured one by one.

For soil culture, sterile seeds were first sown on MS media (supplemented with 1% sucrose and 6 g L^–1 agar), after which they were placed in a 4°C refrigerator for three days to ensure uniform germination and allowed to grow at 22°C under long days (16 h light/8 h dark photoperiod) for 7 days. Then, the seedlings were transplanted into soil, with one seedling per pot. After 14 days of growth, images were taken, and rosette leaves and root lengths were measured separately.

Auxin contents were measured by the indoleacetic acid (IAA) and auxin enzyme-linked immunosorbent assay (ELISA kit, RXJ1401005PL, Quanzhou Ruixin Biological Technology Co., LTD) kits following the manufacturer’s protocol. For sample collection, the samples were ground in 1 mL of 80% (v/v) methanol. The extract was then centrifuged at 8000 rpm for 1 h; Afterward, the supernatant was passed through a C-18 column, and the details of the steps were as follows: washing with 80% (v/v) methanol, 100% (w/v) methanol, 100% (w/v) ether and then 100% (w/v) methanol. Afterward, the hormone fractions were dried and dissolved in 1 mL of phosphate-buffered saline (PBS; pH 7.4) for further analysis. After they were mixed, the samples were incubated at room temperature for 30 min and then centrifuged at 8000 rpm for 15 min at 4°C. The supernatant was then removed and stored temporarily at 4°C for later use.

For determination, the proteins were quantified using a protein quantification kit (RXKM0488-100, Quanzhou Ruixin Biological Technology Co., LTD) to facilitate the subsequent auxin content calculation. The determination of auxin was as follows. First, all the reagents were prepared, after which they were incubated at room temperature for 30 min. Washing solution was prepared according to the sample amount to be measured. A precoated plate was then taken, and a blank control without any liquid was also included. Two wells were designated for each 50 μL of calibration material, and 50 μL of sample material to be measured was to each other test well. Fifty microliters of biotinylated antigen were subsequently added to all the wells except the blank control well, and the contents of the wells were thoroughly mixed. The plates were then sealed with a membrane and incubated at 37°C for 60 min. For manual washing of the plates, the liquid in the wells was first discarded, after which each well was filled with washing liquid. Then plants were subsequently incubated at room temperature for 10 s, dried by swinging (which was repeated 3 times) and then patted dry. Chromogenic agent A (50 μL) and chromogenic agent B (50 μL) were added to each well. Afterward, the plates were shaken and the contents of the wells were thoroughly mixed; the plants were subsequently incubated at 37°C in the dark for 15 min. Then, 50 μL of stop solution was added to each well. A microplate reader was used to determine the absorbance at 450 nm in each well. Then, a standard curve equation was generated, with the standard concentration representing the horizontal coordinate and the corresponding absorbance (OD value) representing the vertical coordinate, using computer software. Four-parameter logistic curve fitting (4-pl), the sample absorbance (OD value), and the standard equation were then to calculate the sample concentration value. Three biological replicates of each hormone were included for this experiment.

Total RNA was extracted from the roots of vertically cultured 14-day-old seedlings using TaKaRa RNAiso Plus (9109, Takara). Two micrograms of RNA were used for library construction; each sample was replicated three times. The transcriptomic data set used in this study was obtained using the Illumina HiSeq platform, and 150-bp high-quality trimmed paired-end reads were generated. The trimmed reads were subsequently mapped to the reference genome sequence of Arabidopsis using HISAT2 (http://ccb.jhu.edu/software/hisat2/faq.shtml) with the default settings (Kim et al., 2015). Differentially expressed genes (DEGs) were analyzed using edgeR (http://bioinf.wehi.edu.au/edgeR/) (Smyth, 2010). Reads of the RNA-seq data were mapped to the Arabidopsis reference genome (The Arabidopsis Information Resource 10 (TAIR10)), and genes whose expression was more than twofold that of the WT (P < 0.05) were considered differentially expressed.

MS519 used in the experiment was a total nitrogen medium containing ammonium and nitrate nitrogen, while MS531 was a nitrogen free medium without any form of nitrogen. Sterile seeds of WT plants, tppi1 mutants and OE5 overexpression lines were sown vertically on MS media (MS519, Phytotech, USA; supplemented with 0.5% sucrose and 10 g L^–1 agar) and MS nitrate media with different concentration of KNO₃ (MS531, Phytotech USA; supplemented with 0, 10 μM, 100 μM, 1000 μM and 10 mM KNO₃, 0.5% sucrose and 10 g L^–1 agar). Then, the plates were all placed in a plant tissue culture chamber, and 12 or 14 days later, images were taken. The primary roots and lateral roots were then removed and measured one by one.

Our previous study found that, compared with the WT, the tppi1 mutant has a significantly shorter primary root (Lin et al., 2020). To verify whether this phenotype is caused by the AtTPPI gene, we introduced the same AtTPPI overexpression construct in which the gene was driven by the CaMV 35S promoter mentioned previously into the tppi1 mutant. T3 homozygous lines were used for further root length assays. The results showed that the root length of the complementary lines (Com2, Com23) could be restored to that of the WT on the vertical MS media ( Figure 1 ). Statistical analysis further suggested that the short-root phenotype of the tppi1 mutant was indeed caused by the AtTPPI gene ( Figure 1B ).

We performed the same experiment in soil. One-week-old seedlings were transferred to 6 cm×6 cm pots filled with soil, and the phenotypes of the plants after 2 weeks of growth were imaged. Similar results were obtained as those above in MS culture ( Figure 1C ), and these were further confirmed via statistical analysis of the primary root length ( Figure 1D ). In addition, we measured the rosette leaf dimeter and statistically analyzed the data. The results showed that the rosette leaf size of Com2 and Com23 could also be restored to that of the WT ( Figure 1E ). This indicates that both the short-root phenotype and the small rosette size of the tppi1 mutant were caused by the AtTPPI gene.

The GFP fluorescent signal of the DR5rev:GFP marker in the root tips of tppi1 mutant and WT plants verified that the tppi1 mutant has lower auxin contents than the WT plants do (Lin et al., 2020). To investigate the function of AtTPPI in terms of auxin, we further measured the auxin levels of WT plants, the tppi1 mutant and overexpression line OE5 with an ELISA kit for auxin. The results showed that the overexpression line OE5 had a significantly increased auxin level compared with that of the WT ( Figure 2A ). In addition, the auxin level of the tppi1 mutant was significantly lower than that of the WT, the results of which were consistent with the weaker DR5rev:GFP fluorescent signal in the mutant (Lin et al., 2020).

Meanwhile, we also crossed the tppi1 mutant and overexpression line OE5 with DR5:GUS marker plants that showing auxin levels, respectively. The expression level of DR5:GUS in OE5 root was significantly higher than WT plants and tppi1 mutant ( Figure 2B ). The result is similar with the above auxin content determination, which further indicate that the elevated AtTPPI expression indeed increase the auxin level.

To further evaluate the role of AtTPPI in modulating the plant auxin response on a broader scale, we analyzed the transcriptomes of WT plants and the tppi1 mutant. Total RNA of roots from 2-week-old seedlings growing vertically on MS media was isolated and subjected to RNA-seq (for which three biological replicates were included). A total of 3 Gb of clean data were obtained, which was mapped to the sequences of the gene models in the TAIR10 assembly (Smyth, 2010). The fragments per kilobase of transcript per million (FPKM) value of each gene were calculated for the WT and tppi1 mutants. By using pairwise comparisons of the tppi1 mutants and WT plants, we defined DEGs as those whose FPKM values changed by at least twofold (p value < 0.05) (Smyth, 2010). We then performed a comparative analysis of the WT and tppi1 mutants. According to the screening conditions, 906 DEGs were identified; among these DEGs (tppi1 vs. WT), 538 were upregulated, and 368 were downregulated ( Figure 3 ). We expected that some of these genes would contribute to the significantly decreased primary root length of the tppi1 mutants.

We therefore performed a gene functional annotation analysis of the 538 upregulated and 368 downregulated genes ( Supplementary Table S1 ). Gene Ontology (GO) analysis revealed that the 538 and 368 genes were involved in various biological processes and molecular functions. We mainly focused on the DEGs related to the auxin response and found some important genes, including 7 upregulated genes and 2 downregulated ones ( Figure 3 ). These important genes were reported to be involved in various processes in response to auxin. Interestingly, among the upregulated genes, there were three GH3 family genes identified, namely, GH3.9, GH3.12 and GH3.3. Research on the function of GH3 genes, which are primary auxin response gene family members, has mainly focused on auxin, including interactions with auxin and auxin response factors and interactions with plant defense responses mediated by plant signaling molecules such as SA (Staswick, 2005; Park et al., 2007; Wang et al., 2011; Weiste and Dröge-Laser, 2014). These genes are mainly involved in auxin inactivation (Staswick, 2005). Loss of function of the GH3.9 gene has been reported to result in longer primary roots of Arabidopsis (Khan and Stone, 2007). Interestingly, the increased fold-change of GH3.9 expression was the highest. The increased expression levels of the GH3.9 gene in the tppi1 mutant may contribute to the significantly shorter primary roots ( Figure 1 ). In addition, apart from GH3.9, GH3.12 has been reported to function in the defensive response, and loss of function of GH3.12 resulted in decreased defense against pathogens, which suggested that the AtTPPI gene might function in plant defense responses.

In addition, through analyzing the Top10 GO terms of ONTOLOGYs of DEGs, we found that among the upregulated DEGs, “S-glycoside metabolic process, Glycosinolate metabolic process, Glucosinolate metabolic process and Photosynthesis, light reaction” were enriched ( Figure 4 ), which indicate that the phenotype of tppi1 mutant is likely connected with these processes. Our recent study also partly proved that sugar metabolism of tppi1 mutant indeed changed, including increased sucrose, starch level and decreased glucose, which is likely responsible for the various development phenotype of tppi1 mutant. Meanwhile, the molecular function responsible for the tetrapyrrole binding accounted for the most ( Figure 4 ). Tetrapyrroles are linear or cyclic molecules containing four pyrrole rings, they usually function as cofactors in plants (Warren and Smith, 2009). In plants, apart from play role in photosynthesis and respiration, tetrapyrroles also participate in the assimilation of nitrogen/sulfur (Luo et al., 2020). Also, tetrapyrroles and tetrapyrrole-containing proteins have been reported to mediate the cellular detoxification of reactive oxygen species (Busch and Montgomery, 2015). The abundance of these genes suggests that AtTPPI may play a role in detoxification and nitrogen assimilation.

In addition, we noted that metabolism-related genes including secondary metabolic process and sulfur compound metabolic process accounted for a large proportion of down-regulated DEGs ( Figure 5 ), which preliminarily suggested that some metabolic pathways might be inhibited in the tppi1 mutants. This is exactly consistent with the growth and development phenotype of tppi1 mutant. For instance, tppi1 mutant had significantly reduced rosette leaves, late-flowering, stunted root length, and numerous root hairs, shorten hypocotyl and delayed seed germination. In future, we will do more research about the relationship between the DEGs and the respective phenotype.

Previous studies have suggested that LONG HYPOCOTYL 5 (HY5) plays a role in suppressing auxin signaling (Sibout et al., 2006; Cluis et al., 2004). HY5 is a basic domain leucine/zipper (bZIP) TF that is an important regulator that promotes photomorphogenesis and participates in the growth of plant roots, and this protein is regulated by light and hormones (Oyama et al., 1997; Toledo-Ortiz et al., 2014; Nawkar et al., 2017). Previous chromatin immunoprecipitation (ChIP) coupled to DNA chip hybridization (ChIP-chip) results also found that AtTPPI is an in vivo target of HY5 (Lee et al., 2007). Apart from its function in modulating auxin levels, HY5 is also involved in nitrogen metabolism and works to integrate the light-mediated nitrogen regulatory network (Chen et al., 2016; Gelderen et al., 2021). Nitrogen is an essential element in plant nutrition. Moreover, plants will preferentially absorb nitrate nitrogen (NO₃ ^–) from the soil. Studies have shown that both the TF HY5 and its homolog HYH may directly induce the expression of the light-mediated high-affinity nitrate transporter gene NRT2.1 and further activate nitrate transporters by promoting sucrose production and efflux (Chen et al., 2016). In view of this, we speculated whether AtTPPI could regulate primary root growth at high concentrations of nitrate.

Above transcriptome data also found that, there were two downregulated genes, DEG18 and AIR1 ( Figure 3B ). DEG18 (which is also named Araport11) is an auxin-responsive family protein-encoding gene that may function in lateral root formation and be activated by OXS2 under salt treatment (Neuteboom et al., 1999; Jing et al., 2019). AIR1 is an auxin-induced gene and may also participate in lateral root formation (Neuteboom et al., 1999). Therefore, we further explore the lateral root phenotypes of the tppi1 mutant and WT plants. The results showed that after 14 days of growth on nitrogen free medium with 10 mM KNO₃ (MS531+10 mM KNO₃), the OE5 plants still had significantly longer primary roots than the WT plants and tppi1 mutants did ( Figure 6B ). On the control MS media, the primary root length of the OE plants was not different from that of the WT ( Figure 6A ). Further statistical analysis confirmed the above results ( Figure 6C ). In addition, we found that the number of lateral root number of OE5 was significantly more than the WT plant under high nitrate conditions ( Figure 6D ), and the tppi1 mutant has less lateral root number than WT plant. The statistical analysis also proved that ( Figure 6D ). Different from the results of primary root growth, under the total nitrogen medium (MS519), the OE5 plants also has significantly more lateral root number ( Figure 6D ), while the tppi1 mutant has same phenotype with the WT plants.

To further evaluate the effluence of AtTPPI on lateral root in detail, we performed the root assay under nitrogen free medium with different concentration of nitrate including 0, 10 μM, 100 μM and 1000 μM KNO₃. The results showed that the primary root length of the tppi1 mutant was significantly inhibited at all concentrations ( Figures 7A, B ). OE5 overexpression plants significantly increased the primary root length at the other three concentrations except for the 10 μM KNO₃ condition ( Figures 7A, B ). These results indicated that the application of KNO₃ promoted the growth of primary roots. At the same time, the number and length of lateral roots were counted. The results showed that at 0, 10, 100 μM, the average lateral root number of the tppi1 mutant was significantly lower than that of the wild-type plant, while at 1000 μM, the average lateral root number of the tppi1 mutant and OE5 was higher than that of the wild-type plant ( Figure 7C ). In addition, at the concentration of 100 and 1000 μM KNO₃, the long lateral root number of OE5 plants was significantly higher than the tppi1 mutants and WT plants ( Figure 7D ). Combined the above results, it can be seen that exogenous potassium nitrate can promote not only the growth of primary root, but also the growth and elongation of lateral root.