The Mechanisms Of Red Light-induced Systemic Regulated Photosynthetic Induction Of Tomato

Improving photosynthesis efficiency is one of key factors for increasing crops yield.The rate of photosynthetic induction is critical for plant to make best use of dynamic light and the important factor that affects the entire plant photosynthesis. Systemic sigalling is an important regulation mechanism for plant growth, development and stress response. Plant photosynthesis and photosynthetic induction are also involved in systemic regulation.However, light induced systemic regulation of photosynthetic induction remains underexplored. In this study, tomato (Solarium lycopersicum) was used as the material to investigate the physiology and molecular mechanism of light induced systemic regulation of photosynthetic induction, from auxin signal, H2O2 signal and cyclic election flow(CEF)through grafting, virus induced gene silencing technology (VIGS) and the RNA interference technology. The results are as follows:1. We have studied the characteristic of systemic regulation of photosynthetic induction in tomato leaves.To verify whether systemic signalling is involed in photosynthetic induction of tomato leaves, we marked tomato leaves from NO.1 to NO.6 by their sequence of expansion, taking NO.4 as the target leaves.We subjected the apex, upper expanded No.5and No.6 leaves between the apex and the NO.4 leaves, and the NO.lto NO.3 leaves of tomato to a 30 min of white light at 300 μmolm-2s-1 entire darkness as control,and then determined the changes in CO2 assimilation at target leaves at 1500 μmolm-2s-1 photosynthetic photon flux density (PPFD) for 30 min. As compared to the dark control, exposure of the apex to white light for 30 min resulted in a faster induction of CO2 assimilation in the target leaves and the time to reach 50% (T50) and 90%(T90) of the saturated CO2 assimilation decreased from 8.17 min to 4.30 min and from 19.7 min to 14.7 min, respectively. However, pre-illumination of the upper expanded leaves or the down leaves did not change T50 and T90. These findings demonstrated that only the light-exposed apex could send systemic signal(s) to the target leaves for the induction of CO2 assimilation in response to the light stimuli.To determine how light systemically induce CO2 assimilation in the down remote leaves, we applied light at 300 μmolm-2s-1 with different wavelengths to the young shoot for 30 min prior to the measurement of CO2 assimilation in the target leaves. Among the light spectrum examined, red light (660 nm) enhanced whilst far red light (735nm) delayed the induction of CO2 assimilation of target leaves.Other lights:blue, yellow, green, cyan, purple, orange, however, did not alter the induction of CO2 assimilation, T50 and T90. We grafted the young shoots of wild type (WT), mutants deficient in phytochrome A (phyA), phytochrome Bland B2 (phyB1B2) and cryptochrome 1 (cry1)with two developing leaves onto stems of wild type plants with four leaves, resulting in four grafting combinations, WT/WT, phyA/WT,phyB1B2/WT and cry1/WT, respectively. A pre-illumination of white light for 30 min resulted in an earlier induction of CO2 assimilation in the stock leaves (NO.4 leaf) of WT/WT, phyA/WT and cry1/WT plants as compared to the dark control. However, white light-induced CO2 assimilation was compromised in phyB1B2/WT plants with little changes in T50 and T90 although it was measured in the leaves of WT, demonstrating that phyB in the apex is required for the systemic induction of photosynthesis in response to white light.2. We have studied the role of IAA in light-systemic regulated photosynthetic induction of tomato leaves.Pre-illumination with white light induced transcript of flavinmonooxygenase (FZY) (a step-limiting gene in IAA biosynthesis) and PIN1(a marker gene for PAT),and the accumulation of IAA in the apex and the target (4th) leaves. Such increases were also found in the apex and the target leaves of phyA/WT and cry1/WT plants. In contrast, white light failed to induce the transcript of FZY and PIN1, so is also true for the IAA accumulation in the apex and the target leaves in the phyB1B2/WT plants. In agreement with the increased abundance of IAA accumulation, white light induced the transcript of IAA15 (a marker gene of IAA signalling) in the target leaves of WT/WT, phyA/WT and cry1/WT plants, but not in the same rootstock leaves of the phyB1B2/WT plants.Top lighting with white light and red light induced DR5::GUS intensity as soon as 30 min after the exposure. In contrast, top lighting with far red light markedly decreased DR5::GUS intensity whilst blue light did not alter the intensity in the roots. Similarly, auxin signaling was induced in the roots of WTIDR5::GUS, phyA/DR5::GUS and cryl/DR5::GUS tomato DR5::GUS grafting plants, but not in the roots of the phyB1B2/DR5::GUS plants by the pre-illumination.All these results indicated that phyB induced not only an increased IAA biosynthesis in the apex but also auxin signaling in down leaves and roots. Similar to the pre-illumination, application of IAA accelerated the induction of CO2 assimilation in the down leaves under dark condition. However, application of N-1-naphthylphthalamicacid (NPA, an inhibitor for PAT) abolished white light-induced CO2 assimilation in the target leaves. Therefore, IAA synthesized in the apex may function as a systemic signal to induce CO2 assimilation in the down leaves. White light-induced CO2 assimilation was abolished in the WT leaves of the dgt/WT (dgt, an auxin insensitive mutant) grafting plants,T50 and T90 did not change with the pre-illumination.In addition, this chapter also indicated that under the normal temperature the environment R:FR ratio changes had no significant effects on the ABA and JA content of tomato leaves.3. We have studied the role of H2O2 in light-systemic regulated photosynthetic induction of tomato leaves.Chemical and qRT-PCR analysis revealed that the preillumination of the young shoots triggered the transcript of RESPIRATORY BURST OXIDASE HOMOLOG 1 (RBOH1), which encodes NADPH oxidase to generate H2O2 in the apoplast, and induced an accumulation of H2O2 in the target leaves of WT/WT, phyA/WT and cry1/WT plants, but not in the WT target leaves of the phyB1B2/WT plants by the pre-illumination.Using a CeCl3-based procedure, we found that pre-illumination induced H2O2 accumulation in the cell walls of mesophyll cells that face intercellular spaces. However, light-induced RBOH1 transcript and apoplastic H2O2 accumulation was abolsihed in the target leaves of dgt/WT plants. These results suggested that top lighting-activated apoplastic H2O2 production was dependent on auxin signaling in these plants.To determine the role of RBOH1 in pre-illumination-induced CO2 assimilation in the target leaves, we generated RBOH1-RNAi plants (rbohl) and grafted onto WT plants as stock. We found that pre-illumination-induced CO2 assimilation was compromised in the target leaves of WT/rbohl plants and the same was also true for rbohl/rbohl plants. All these results indicated that auxin-induced H2O2 production in the target leaves played a critical role in the pre-illumination-induced CO2 assimilation.4. We have studied the role of cyclic electron flow (CEF) in light-systemic regulated photosynthetic induction of tomato leaves.To examine how pre-illumination-induced H2O2 was involved in the induction of CO2 assimilation, we compared CEF in target leaves of WT/WT, WT/rbohl and rbohl/rbohl plants. Under the dark condition for the leaves, CEF was very low in all these plants. However, pre-illumination of the young shoots significantly induced CEF in the target leaves of WT/WT plants and this induction was not observed in the target leaves of WT/rbohl and rbohl/rbohl plants. Meanwhile, this induction of CEF was also absent in the target leaves of phyB1B2/WT plants and dgt/WT plants but could be found in the target leaves of phyA/WT and cry1/WT plants. Therefore, pre-illumination of the young shoots induced CEF is dependent on H2O2 production by RBOH1 NADPH oxidase in the target leaves. CEF promotes CO2 assimilation through ATP generation. In agreement with the increased CEF, there was a 47.2%-57.7% increase in the ATP content in the WT leaves of WT/WT, phyA/WT and cry1/WT. As the null response of CEF to light, top lighting-induced increase in ATP production was not found in the WT leaves of phyB1B2/WT, dgt/WT, WT/rbohl and rbohl/rbohl plants. We then determined whether the induced CEF is essential for the light-induced photosynthetic induction. ORR, which links plastid NAD(P)H dehydrogenase complex activity was previously found to be involved in the CEF.Virus-induced silencing of ORR (pTRV-ORR) resulted in a reduction of ORR transcript by 68.7%as compared to the empty vector plants (pTRV). pTRV-ORR plants showed null response to the pre-illumination in term of CO2 assimilation induction, CEF and ATP.However, pre-illumination induced H2O2 accumulation in the target leaves of both pTRV and pTRV-ORR plants. Therefore, H2O2-dependent CEF is essential for the apex induced photosynthesis in response to the top lighting stimuli.