Chilling tolerance of Dular and Lsi1-OX rice
The Dular and Lsi1-OX transgenic lines presented different tolerances to the chilling treatment of 4 °C for 36 h. Most of the leaves from the Dular rice became whiter, whilst the Lsi1-OX line maintained tolerance to chilling (Fig. 1A). Further studies showed that the Lsi1 overexpression vector was driven by the ubiquitin promoter, and the translate OsNIP2;1 was localised in the cytoplasm (Fig. 1B), which differs significantly from the original localisation in the cell membrane (Ma et al. 2006). This result suggests that OsNIP2;1 may have more roles than its initial function, which belongs to the aquaporin family and controls silicon accumulation in rice . SEM images of the leaves revealed that silica bodies in the Lsi1-OX rice leaves were bigger than those of the Dular rice (Fig. 1C).
Subcellular localisation of OsSHMT
Overexpression of Lsi1 in Dular rice results in changes in the expression of thousands of genes , including the OsSHMT gene. Expression of OsSHMT was up-regulated in the Lsi1-OX rice in comparison with the Dular rice (Fig. 2A). Subcellular localisation of OsSHMT (LOC_Os03g52840) in the rice protoplast then showed that yellow fluorescence was concentrated around the nucleus, and no obvious endonuclear fluorescence was observed, whereas significant yellow fluorescence was seen in the whole nucleus in the rice protoplast transformed with the eYFP vector (Fig. S1). This suggested that OsSHMT was localised on the endoplasmic reticulum around the nucleus. Further co-localisation of the endoplasmic reticulum marker protein with mcherry indicated that OsSHMT was widely distributed in the endoplasmic reticulum, as the mcherry fluorescence from the marker protein and yellow fluorescence from OsSHMT infused with eYFP was completely overlapping (Fig. 2B). OsSHMT is involved in the photorespiratory processes of plants, and existing studies have suggested that the protein is localised to mitochondria, chloroplasts and cytoplasm. The results of this study complement those findings.
Promoter region of OsSHMT and the binding proteins
The promoter region 2597 bp upstream of the CDS of the OsSHMT gene from Dular rice was amplified and labelled with biotin at the 5′ flanking region of this DNA fragment (Table S1). According to the DNA pull-down results, compared with the control group at room temperature, chilling treatment of Lsi1-OX induced more proteins to bind to the promoter region of OsSHMT. In contrast, chilling treatment of the Dular rice had no significant effect on the proteins binding to the promoter (Fig. 3A). Identification of the proteins showed that retrotransposon protein (Ty3-gypsy subclass), glyceraldehyde-3-phosphate dehydrogenase, and AT hook motif protein were identified from both the Lsi1-OX and Dular rice under chilling treatment or room temperature conditions; however, some proteins, such as histone H1,nucleic acid binding protein (NABP) and tubulin/FtsZ domain-containing protein (LOC_Os03g51600.1) were only identified from the chilling-treated Lsi1-OX group; another tubulin/FtsZ domain-containing protein (LOC_Os05g34170.2) was identified from the chilling-treated Lsi1-OX and Dular groups and from the Dular group at room temperature. AAA-type ATPase family protein was identified from the Dular group at room temperature but not from the chilling-treated Dular group, and this protein was induced in the chilling-treated Lsi1-OX group in comparison with its control group at room temperature (Table 1). The tubulin/FtsZ domain-containing proteins (LOC_Os03g51600.1, LOC_Os05g34170.2, LOC_Os07g38730.1), histone H1 and NABP were selected to analysis their gene expression level on the two rice. A comparison of the gene transcription levels in Dular and Lsi1-OX rice after chilling treatment for 12, 24 and 36 h in comparison with 0 h, revealed opposite trends in the two rice lines, and the gene expression level was up-regulated in the Lsi1-OX rice in comparison with Dular rice under the same treatment conditions (Fig. 3D). The results indicate that these genes act in combination to exert a positive role in the regulation of OsSHMT expression.
Proteins interacting with OsSHMT
Based on the GFP-TRAP method to obtain the proteins interacting with OsSHMT, it was found that several proteins co-precipitated with OsSHMT, in comparison with the GFP-vector control (Fig. 4; Table S2). Some of these proteins, including ATP synthase α subunit, ATP synthase β subunit, heat shock protein 70, mitochondrial substrate carrying family protein E, ascorbate peroxidase 1, are defense proteins that interact with OsSHMT protein (Table 2). Further determination of the interaction of OsSHMT with ATP synthase subunit α, ATP synthase subunit β, Hsp70, MSCP and APX from the rice showed that yellow fluorescence was detected in the leaves of tobacco infected with OsSHMT and each of these proteins respectively. No fluorescence was detected in the control group, demonstrating that OsSHMT positively interacts with the above proteins. The results indicate that OsSHMT interacted with defence-related proteins in rice, including APX, MSCP, HSP70 and ATP synthase, to jointly regulate chilling resistance.
H2O2 content in the OsSHMT transgenic A. thaliana and wild type
To further indicate the function of OsSHMT in scavenging H2O2, wild-type A. thaliana and positive transgenic T3 A. thaliana seedlings were exposed to a temperature of 4 °C for 12, 24 and 36 h. The leaves of the wild type showed more reddish-brown spots after DAB staining. The transgenic line also showed reddish-brown spots, but the spots were small in size and number. Determination of the leaf H2O2 content of the transgenic line and wild type of A. thaliana showed that the increase in H2O2 in the transgenic line of A. thaliana after chilling treatment was significantly lower than that of the wild type that underwent the same treatment.
OsNIP2;1 interacts with ATP synthase subunit β from rice
Lsi1 encodes the aquaporins protein OsNIP2;1, which is a nodulin 26-like intrinsic protein (NIP) and is localised in the membrane of the cell. Overexpression of Lsi1 in Dular rice using a ubiquitin promoter resulted in the cytoplasm localisation of this protein, enabling OsNIP2;1 to play multiple roles. The interaction between OsNIP2;1 and OsSHMT was also investigated. The results showed no direct interaction between OsNIR2;1 and OsSHMT. However, OsNIP2;1 interacted with ATPsyn-β, a protein that also interacts with OsSHMT, and the results indicate that ATPase-β acts as an intermediate junction between OsNIP2;1 and OsSHMT.