Proteomic analysis of RTN13 knockout

To investigate any subtle (or un-tested) changes withinArabidopsisseeds which lack the expression of RTN13, total protein extracts were prepared from wild type and

rtn13knockout seeds and subjected to 2-dimentional SDS-PAGE and Difference In- Gel Electrophoresis (DIGE). However, preliminary analysis of the protein indicated that there was no difference between the two samples (data not shown). However, it has been reported that after protein extraction and resolution, not all desired proteins can be visualised, and methods of extraction vary depending on whether membrane or soluble proteins are to be analysed (Isaacsonet al., 2006).

0 1 2 3 4 5 6 7 wt 1 KO 1 OE 1 wt 2 KO 2 OE 2 Fa tt y A ci ds µg /s ee d

Figure 6.9 Lipid analysis of wild type, rtn13 knockout and YFP-RTN13 over-expressing Arabidopsis seeds. Total fatty acid content of Arabidopsis

seeds were analysed using gas chromatography. rtn13 knockout and YFP- RTN13 over-expressing seeds were compared with wild type seeds in two geographical locations (1-2). T-tests showing statistical significance against wild type seeds are shown.

n 10 10 10 10 9 10 t-test 0.9323 0.0014 0.4763 0.2630 Significant No Yes No No Change (µg) +0.016 +0.684 -0.160 +0.290

6.7 Discussion

The previously described 35S-driven YFP-RTN13 localises to the ER inArabidopsis

embryos, roots and leaves. In roots and leaves, constriction of the tubular ER was observed when RTN13 was expressed in anArabidopsisline transformed with GFP- HDEL. In embryos, RTN13 localises to the ER, is excluded from the protein storage vacuoles and co-localises with GFP-HDEL in imbibed seeds.

Wild type and GFP-HDEL Arabidopsis lines were transformed with 35S:YFP- RTN13 in order to determine the localisation of RTN13 within its native organism and compare any phenotype observed with expression in tobacco. Embryonic, root and leaf cells were examined. YFP-RTN13 localised to the ER, similar to that in tobacco and in root and leaf cells, RTN13 over-expression induced the formation of constrictions in tubular ER. This had already been observed when YFP-RTN13 was biolistically delivered intoArabidopsis(Figure 4.16) and shows that the formation of constrictions is not a species-specific phenomenon. YFP-RTN13 localised to the ER in the embryo and co-localised with GFP-HDEL in imbibed seeds, although constrictions were not observed at this stage of development.

RTN13 under control of its native promoter is expressed in the late stages of embryo maturation and localises to the ER. Expression ceases in the mature embryo and imbibed seed, which is consistent with the expression levels based on transcript levels using eFP Browser (Winteret al., 2007).

In order to determine the tissue-specific expression pattern of RTN13, the genomic sequence of RTN13 was expressed under its native promoter with YFP fused at the C-terminus. A heterozygous transgenic line was created and the expression of RTN13 examined. mRNA transcript levels illustrated in eFP Browser indicated that expression of RTN13 occurs in the early curled-cotyledon, and peaking in the late green-cotyledon stages. High transcript levels are also detected in the dry seed and are reduced in the 24 h imbibed seed. There is no expression of RTN13 in any other tissue of the plant. The expression of native RTN13 in Arabidopsis was intended to validate these mRNA expression levels, and determine the localisation of RTN13 at native expression levels. Ageing siliques from three different stages of development

were selected and expression levels compared with dry and imbibed seeds. Embryos were dissected and subjected to confocal imaging to establish the expression of RTN13. Low levels of expression were observed at the early curled-cotyledon stage; mid-levels of expression were evident at the late curled/early green-cotyledon stage and the highest expression was observed at the late green-cotyledon stage (Figure 6.4). RTN13 labelled the ER in the embryo at all stages. However, when examining either dry or imbibed seeds, although mRNA transcripts were detected at these stages (eFP Browser), fluorescence had diminished in the embryo, whereby small punctae and the PSVs were now weakly fluorescent. This may indicate that the protein is turned over upon seed dehydration.

These data indicate that both the over-expression and native expression of RTN13 result in localisation to the ER, and under the control of its native promoter, RTN13 is specifically expressed during the late stages of embryo maturation, and ceases in the fully mature, dry seed. Although RTN13 is the only reticulon to be expressed exclusively in seeds, RTN1 and RTN2 are also expressed in the seed (see Appendix Figure A8). Presumably, the formation and maintenance of ER tubules may be upheld by the expression of all three isoforms, as they display similar properties. However, the N-termini, thought to carry out the functional, rather than structural, role of reticulons (Iwahashiet al., 2007), vary between these isoforms. Therefore, the N-terminus of RTN13, although one of the shortest of all Arabidopsis reticulons, may possess a functional role within cells of the developing embryo. To elucidate this functional role, an Arabidopsis T-DNA insertional rtn13 knockout line was obtained and characterised.

The effects of knocking out RTN13 were investigated using a homozygous T-DNA insertion line which was confirmed by RT-PCR. Seed germination, protein trafficking to the vacuole and ER, vacuole and lipid body morphology and lipid levels in the seed were analysed. However, no obvious phenotypic differences were detected between wild type and knockout plants.

A homozygous T-DNA insertional knockout of RTN13 was obtained (from NASC) and the position of insertion was confirmed in the first exon of RTN13. Diminished expression levels were then established by RT-PCR. Expression Angler software (Toufighi et al., 2005) generated a list of candidate genes expressed with a similar

pattern to RTN13. Of the 25 genes suggested, the most notable were HVA22 (a homologue of the reticulon-like DP1 and Yop1p genes); oleosin 1 and 2; andαTIP, a tonoplast intrinsic protein functioning as a water uptake channel in the vacuolar membrane. Based on these related genes and potential interacting partners, several characteristics of seeds were compared between wild type, knockout and the over- expressing RTN13 lines.

The rate of germination and the processing of storage protein precursors were investigated, although no difference was observed between the three lines. The morphology of the ER, storage vacuoles and lipid bodies were examined using DiOC6, auto-fluorescence and Nile Red, respectively. Similarly, no difference was observed between the morphology of these organelles. Owing to the timely expression pattern of RTN13 and oleosins, which are incorporated into the lipid monolayer of the lipid bodies in the ER, the three lines were subjected to gas chromatography-based lipid analysis from two sets of samples grown independently. However, no significant difference was evident between the samples. Total proteins extracted from wild type and knockout seeds were subjected to 2D-gel DIGE analysis, though preliminary results suggested no difference between the two samples.

The fact that no phenotype was observed in the rtn13 knockout may indicate that other reticulon isoforms present in the seed can compensate for its loss. Owing to its short N- and C-termini, RTN13 may act solely as a structural protein helping shape ER tubules; a role which can be compensated for by RTN1 and RTN2. Accordingly, RTN1, 2 and 4 have been shown to interact with each other through yeast two hybrid (Hwang & Gelvin, 2004) and recently, it has been shown that RTN13 can homo- and hetero-dimerise with RTN3 and RTN4 (Sparkeset al., 2010) The expression patterns of many of theArabidopsisreticulons overlap throughout the tissues of the plant and may indicate a relatively high degree of redundancy within the reticulon gene family.

As suggested previously, however, the tubular morphology of the ER is unlikely to be dependent on one protein alone, and therefore other proteins such as HVA22 and RHD3 may be involved in generating ER tubules. Seed-specific isoforms of these genes are present in Arabidopsis, of which HVA22B is displays a similar expression

pattern to RTN13. Through the regulation of tubules and sheets within the ER at various developmental stages, cellular processes such as the deposition of storage proteins or the formation of lipid bodies can be achieved with the most efficient distribution of ER. Reticulons, in part, may help regulate the balance between sheets and tubules within the cell, whilst also being involved in other cellular processes via interactions with their N-terminal regions; processes which take place near the ER membrane.

Cisternal ER is suggested to be the site of protein synthesis and is often visualised as being studded with ribosomes. In contrast, tubular ER is often described as smooth ER, and is implicated in lipid metabolism and protein export. The extending reach of cortical ER tubules and increased surface area (in relation to sheets) may facilitate the transfer of lipids from the ER to surrounding organelles, and increase the efficiency of protein export from the ER and its subsequent delivery to downstream organelles in the secretory pathway.

As reticulons have been shown to insert preferentially into curved membranes, this mechanism of segregation may be a feature common to many proteins, or at least reticulons may sequester proteins to regions of tubular ER – where they can carry out specific functions. As the reticulon homology domain is seemingly the ‘core’ of the protein – present in all reticulons, then it is plausible that the down-regulation of one isoform may be compensated by others. RTN13 is similar to the mammalian Rtn4c in that they both comprise little more than the RHD. However, in order to induce a morphological change in the ER, proteins other than Rtn4c are required to be down- regulated, namely DP1. In yeast, a deletion of Rtn1p, Rtn2p and Yop1p results in only a moderate growth defect (Voeltz et al., 2006), and as was shown here - knocking out RTN13 has no effect inArabidopsis, questioning the functional role of individual reticulon proteins.