To determine the presence of 10-OPEA and 12-OPDA during seedling growth, we harvested scutella in developing seedlings over a 14 d time course. Ten d post-planting, 10-OPEA accumulation was >30- and 190-fold above that of 10-OPDA and 12-OPDA, respectively, and 40- and 300-fold higher by 14 d . Cell death, as measured by ion leakage, increased throughout seedling growth and corresponded to the progressive accumulation of 10-OPEA , indicating a strong and positive monotonic correlation between 10-OPEA production and programmed cell death . These data suggest that 10-OPEA is developmentally regulated and that it may play a role in scutella senescence. It is also possible that 10-OPEA levels increase in the scutella in response to soil-borne microbes that attack the dying tissue. While many studies have investigated the biosynthesis and function of 13-LOX derived cyclopentenones such as 12-OPDA, surprisingly little is known about the 9-LOX derived jasmonate-like analogs 10-OPEA and 10-OPDA. We recently demonstrated that 10-OPEA is abundantly and predominantly produced during SLB infection and can function both as a direct plant defense and transcriptionally active signal. In comparative analyses between 9- and 13-LOX derived cyclopentenones, we observed a measurable divergence in function with 10-OPEA displaying significantly heightened cell death promoting activity. To better understand aspects of distribution and abundance,macetas cuadradas grandes and to gain insight into additional functions, we investigated the production of 10-OPEA, 10-OPDA, and 12-OPDA in several maize organs under diverse treatments.
In response to SLB-infection, we observed that 10-OPEA is significantly more abundant than 12-OPDA in both inner-whorl leaf and developing stem node/internode tissues. This bias toward 10-OPEA production was somewhat predictable as transcript levels for the candidate 9-LOXs responsible for 10-OPEA biosynthesis are induced to higher levels during SLB infection than the 13-LOX genes that regulate jasmonate production .Differences in accumulation between 10-OPEA and the two 18:3 derived cyclopentenones may also be due to substrate availability, as 18:3 levels are approximately 2-3 fold lower than 18:2 under SLB infection conditions.Interestingly, 12- OPDA is produced at much higher levels than 10-OPEA in FAW-R treated leaves. Although 10-OPEA was previously shown to be inducible in corn ear worm infested inner-whorl tissues,its production does not significantly increase in fully expanded leaves in response to FAW-R , indicating that insect-induced 10-OPEA in leaves may be specific to particular developmental states of leaf tissue. Large differences in cyclopentenone production were observed in other maize organs including silks and roots, as 10-OPEA levels significantly exceeded those of 10- and 12-OPDA. The physiological and ecological role for above as well as below ground organs to accumulate 10-OPEA is likely due to enhanced specificity for activating pathogen defenses. For example, maize silks facilitate pathogenicity for spores of airborne ear rotting pathogens such as Aspergillus flavus and Fusarium verticillioides.Consistent with previous work demonstrating the significance of maize silks in pathogen defense,10-OPEA has strong antimicrobial activity against both A. flavus and F. verticillioides. Current efforts to generate null mutants in the 10-OPEA biosynthesis pathway will allow the functional role of 10-OPEA in maize defense against biotic threats to be tested genetically. This study addresses the production of the 9- and 13-LOX derived cyclopentenones 10-OPEA, 10-OPDA, and 12-OPDA, in multiple organs under different biotic and developmental conditions.
While these findings broaden our understanding of 10-OPEA, 10-OPDA, and 12-OPDA abundance, distribution, and function, many questions remain. For example, what are the biological functions of 10-OPEA and 10-OPDA in direct defense and defense signaling against a broad range of pathogens and insects? And, what is the occurrence and function of 10-OPEA and 10-OPDA in other monocot and dicot species? Critical to these questions will be the development of effective genetic tools that eliminate or overproduce these metabolites in planta.Plastids originate from a single endosymbiontic event involving a cyanobacterium-related organism. In the course of endosymbiosis a massive gene transfer occurred, during which most plastidic genes were transferred to the host cell nucleus. Consequently, today the majority of plastidic proteins must be post-translationally imported back into the organelle. So far, two protein translocation complexes have been characterized in the outer and inner envelope membrane: Toc and Tic. After passing the outer membrane via the Toc translocon, the Tic complex catalyses import across the IE membrane. So far, seven components have been unambiguously described as Tic sub-units: Tic110, Tic62, Tic55, Tic40, Tic32, Tic22 and Tic20 . Tic110 is the largest, most abundant and best studied Tic component. It contains two hydrophobic transmembrane-helices at its N-terminus, anchoring the protein in the membrane, and four amphipathic a-helices in the large C-terminal domain that areresponsible for channel formation. At the intermembrane space side, Tic110 contacts the Toc machinery and recognizes preproteins. Moreover, loops facing the stroma provide a transit peptide docking site and recruit chaperones such as Cpn60, Hsp93 and Hsp70. Tic110 is expressed in flowers, leaves, stems and root tissues, indicating a role in import in all types of plastids. Heterozygous knockout plants are clearly affected: they have a pale green phenotype, exhibit deffects in plant growth, display strongly reduced amounts of thylakoid membranes and starch granules in chloroplasts, coupled with impaired protein translocation across the IE membrane.
Tic20 is a second candidate within the Tic complex that was proposed to constitute a protein translocation channel. For instance, Tic20 was detected in a cross-link with the Toc complex after in vitro import experiments in pea. In a more recent study, Tic20 was found to form a complex of one megadalton containing a preprotein en route into the plastid after mild solubilization of pea and Arabidopsis chloroplasts, also suggesting its involvement in protein import. Tic20 is predicted to have four a-helical transmembrane domains, and is thus structurally related to mitochondrial inner membrane translocon proteins, namely Tim17 and Tim23 . Distant sequence similarity was also reported between Tic20 and two prokaryotic branched-chain amino acid transporters. Computational predictions place the N- and C-termini in the stroma , however, there is no experimental evidence for the proposed topology in higher plants. The only indication for a Nin-Cin topology is a result of a C-terminal GFP-fusion to a highly divergent member of the Tic20 protein family from Toxoplasma gondii. In the same study, tgtic20 mutants were analysed for protein import into apicoplasts, a plastid type originating from secondary endosymbiosis,frambuesas cultivo and it was found that also this distant homolog of Tic20 is important, albeit probably as an accessory component. The Arabidopsis thaliana genome encodes four Tic20 homologs: AtTic20-I, -II, -IV and -V. AtTic20-I shows the closest homology to Pisum sativum Tic20 . It is present in all plant tissues, and its expression is highest during rapid leaf growth. AtTic20-I antisense plants exhibit a severe pale phenotype, growth deffects and deficiency in plastid function, such as smaller plastids, reduced thylakoids, decreased content of plastidic proteins, and altered import rates of preproteins. Knockouts of AtTic20-I are albino even in the youngest parts of the seedlings. The presence of another closely related Tic20 homolog may prevent attic20-I plants from lethality, since Tic20-IV is upregulated in the mutants. However, additional over expression of AtTic20-IV can only compensate the observed deffects to a very low extent indicating that AtTic20-IV cannot fully substitute for the function of AtTic20-I. Two more distantly related homologs are also present in Arabidopsis . However, their closest orthologs are cyanobacterial proteins, and even though a chloroplast transit peptide is weakly predicted, their localization in the cell remain unknown. Based on structural similarity to channel-forming proteins, cross-links to imported preprotein and protein import deffects detectable in the knockdown mutants, it was hypothesized that Tic20 forms a protein translocation channel in the IE membrane. Furthermore, a cross-link of a minor fraction of Tic110 to Tic20 in a Toc-Tic supercomplex indicates an association of the two proteins. Therefore, it was proposed that the two proteins possibly cooperate in channel formation. However, there was no cross-link detected between the two proteins in the absence of the Toc complex, making a direct or permanent interaction unlikely.Recently, Tic20 was demonstrated to be a component of a one megadalton translocation complex detected on BNPAGE after in vitro import into pea and Arabidopsis chloroplasts. Tic110 could not be observed in this translocation complex, it formed a different, several hundred kilodalton smaller complex, supporting the idea that the two proteins do not associate. However, the expected channel activity of Tic20 has not been demonstrated experimentally yet. In this work we explored the role of Tic20 in relation to Tic110 in more detail.
We analysed the expression of Tic20 in Pisum sativum and Arabidopsis thaliana by quantitative RT-PCR, and compared it directly with the expression of Tic110 in both organisms. Furthermore, semi-quantitative immunoblot analyses revealed the absolute amounts of Tic20 and Tic110 in chloroplast envelopes. Moreover, we showed that Tic20 and Tic110 are not part of a mutual complex in isolated pea IE. After the successful expression and purification of Tic20 we were able to experimentally verify its predicted ahelical structure and Nin-Cin topology. Finally, we report for the first time that Tic20 forms a cation selective channel when reconstituted into liposomes.Due to errors in the annotation of AtTic20-I, currently available Affymetrix micro-arrays do not contain specific oligonucleotides for this isoform and therefore cannot be used to investigate the expression levels of AtTic20-I. We designed specific primers for Tic20 and Tic110 in pea and Arabidopsis and performed a quantitative RT-PCR analysis to obtain comprehensive and more reliable quantitative data about the expression of Tic20 than those available from semi-quantitative analysis and the Massively Parallel Signature Sequencing database. For the analysis, RNA was isolated from leaves and roots of two-week-old pea seedlings as well as four week-old Arabidopsis plants. Arabidopsis was grown hydroponically to provide easy access to root tissue. In all samples, expression of Tic20 was analysed in direct comparison to Tic110 . In pea, expression of both genes was found to be lower in root tissue as compared to leaves. In roots, PsTic110 RNA is 40% more abundant, while in leaves the expression levels of PsTic20 and PsTic110 seem to be in a similar range. In Arabidopsis, AtTic20-I and AtTic110 are expressed to a lower extent in roots than in leaves, similar to pea . These results seemingly contradict those of Hirabayashi et al., who concluded a comparable expression level of Tic20-I in shoots and roots. However, they used a non-quantifiable approach in contrast to our quantitative analysis. Furthermore, in our experiments the overall expression of AtTic20-I and AtTic110 differs notably from that in pea, AtTic110 RNA being about 3.5 and 6 times more abundant than AtTic20-I in leaves and roots, respectively. We also designed specific primers for the second Tic20 homolog in Arabidopsis, AtTic20-IV, and our quantitative method was sufficiently sensitive to precisely define its RNA levels in Arabidopsis leaves and roots, allowing direct comparison with the expression of AtTic20-I and AtTic110 . Transcription of AtTic20-IV had also been investigated in parallel to AtTic110 by Teng et al., who observed a differential ratio of expression using two different methods, of which one was not even sensitive enough to detect AtTic20-IV. A very recent study also investigated the expression of AtTic20-IV, however, without any quantification of their data. Our data show that AtTic20-IV is present in leaves and roots with transcript levels similar to AtTic20-I, but less abundant than AtTic110. Interestingly, and in accordance with the data presented by Hirabayashi et al., transcript levels of AtTic20-IV in roots are higher than those of AtTic20-I, while the opposite is true in leaf tissue. It can be speculated that the observed expression pattern refilects tissue-specific differentiation of both genes. AtTic20-IV may still partially complement for the function of AtTic20-I, as becomes evident from the viability of attic20-I knockout plants and the yellowish phenotype of attic20-I mutants over expressing AtTic20- IV. However, the severe phenotype of attic20-I plants, in conjunction with the observed differential expression pattern, clearly indicates specific functions of the two homologs. Furthermore, a higher AtTic110 expression rate as observed in antisense attic20-I lines might indicate another possible compensatory effect. The expression pattern of the three investigated genes was found to be similar in Arabidopsis growing hydroponically with or without sucrose or on soil . However, gene expression was generally higher in plants growing without sucrose.