Periodic Reporting for period 1 - POSTCAMB (A model for potato storage organ differentiation from the cambial meristem)
Período documentado: 2021-10-15 hasta 2023-10-14
Plant species forming storage organs like bulbs, tubers, or tuberous roots are cultivated as staple food and feed crops because of their high starch caloric content, and elevated yields per hectare. These organs can differentiate from the petioles, stems or the roots, via a secondary growth program whose control is little understood. Tubers and bulbs serve as propagation organs to the plant, remaining dormant in soil during winter, to be reactivated the next spring and generate a new plant that is genetically identical to the mother plant. Plants sense short days and cool nights as an indicator of the approaching winter to induction of these organs. In potato, these external cues induce expression of a member of the FLOWERING LOCUS T (FT) gene family acting as a main tuberigen signal, In this proposal I aimed at characterizing the genes acting downstream of FT in the stolons, and the specific cells that serve as tuber initials. In potato, the SP6A tuberigen signal is produced in the leaves and moves down to the stolon where it interacts with 14-3-3s and transcription factors of the bZIP family, to form a so called Tuberigen Activation Complex (TAC). The downstream targets of the TAC were mostly unknown before this study. We have demonstrated that the vascular cambium serves as initial meristem for tuber formation. The SP6A protein promotes division of these cells and their subsequent differentiation into a storage identity, while a complex regulatory network restricts activation of this program to derivative cells on the phloem side. Outcomes of this work will not only lead to a better comprehension of cambium function, but unveiled key regulators of storage identity thus implying a major breakthrough in our understanding on how these economical relevant organs are differentiated.
Using high throughput cutting edge methods like RNA-Seq, DAP-Seq, ChIP-Seq, and IP-MS, we have identified several novel targets and TAC protein partners at the genome and proteome levels. These extended the very reduced repertoire of direct targets, so far including only SP6A itself and GERMINs. These two genes had been shown to be activated on co-expression of the SP6A and the potato FDL1 bZIP factor. Furthermore, we found that multiple cytokinin pathway genes were directly activated by the TAC, showing that changes in the auxin-CK balance presumably have a pivotal role in inducing the switch of vascular cambium to a storage fate. Interestingly, SP6A had been shown to bind in addition to FDL1 (the potato homolog of FD) other group III bZIP factors, as ABL1 and AREBs. Same as FDL1, these bZIPs include a SAP motif at the C-terminal end that is required for 14.3.3s interaction, and may be implicated in a drought scape tuberization response. Through ChIP-Seq experiments with Hd3a-GFP potato lines (express the rice FT homolog which promotes tuberization), we here showed that the TAC binds the promoter of AGL8/FUL (this gene displays in all RNA-seq studies a co-regulated expression with SP6A), and also APETALA 1 (AP1), CAULIFLOWER (CAL), and SEPALLATA 4 (SEP4), consistently with these MADS-box transcription factors acting as direct TAC targets. Notably, GO term analyses showed that TAC targets were enriched in flowering- as well as cytokinin (CK)-related genes, indicating that CK signaling has a pivotal role in tuber initiation.
Furthermore, we performed DAP-Seq studies on potato genomic DNA by using MBP fusions of the FDL1a, FDL1c, and ABL1 factors expressed in E.coli. As expected, comparison of the FDL1a and ABL1 DAP-Seq peaks identified several binding sites in common with the Hd3a ChIP-Seq, but a significant number of peaks were observed to be unique to Hd3a or the FDL1/ABL1 factors. Peaks unique to Hd3a were found to be enriched in flowering and CK/ JA-related genes, rather than in the salt stress and ABA signaling GO terms as for FDL1a or ABL1. Genes differentially expressed in SP6Aox lines are actually enriched in JA pathway genes, indicating that Hd3a/SP6A activate these targets via complex formation with other regulators distinct to the bZIP factors. This is a fully novel finding that will be further investigated by the host group.
Proteomic studies of chromatin samples of the Hd3a-GFP lines immunopurified using anti-GFP magnetic beads led to the identification of several novel transcription factors that bound the Hd3a FT protein and thus might form alternative transcriptionally active complex with this tuberigen signal. These included a zinc finger factor, TFs of the indeterminate (IDD) family, FRIGIDA-like, GRAS and TCP families. Surprisingly, FDL1 or ABL1 were not included in this protein dataset and therefore parallel studies were carried out with chromatin samples from stolons, to assess whether we were able to detect such bZIPs in these samples and which are currently being analyzed by MS/MS. We cloned the potato IDD, TCP, FRIGIDA-like and GRAS proteins in Y2H vectors to carry out specific protein-protein interaction studies in yeast cells to further analyze their interaction ability with the SP6A, SP5G, BRC1b, FDL1 and ABL1 baits. Our initial Y2H experiments showed very encouraging results which indicated that IDDs, TCPs, and GRAS proteins directly bind various of these tuberization regulators. We are currently verifying these results by using complementary methods like Co-IP.
Furthermore, we performed DAP-Seq studies on potato genomic DNA by using MBP fusions of the FDL1a, FDL1c, and ABL1 factors expressed in E.coli. As expected, comparison of the FDL1a and ABL1 DAP-Seq peaks identified several binding sites in common with the Hd3a ChIP-Seq, but a significant number of peaks were observed to be unique to Hd3a or the FDL1/ABL1 factors. Peaks unique to Hd3a were found to be enriched in flowering and CK/ JA-related genes, rather than in the salt stress and ABA signaling GO terms as for FDL1a or ABL1. Genes differentially expressed in SP6Aox lines are actually enriched in JA pathway genes, indicating that Hd3a/SP6A activate these targets via complex formation with other regulators distinct to the bZIP factors. This is a fully novel finding that will be further investigated by the host group.
Proteomic studies of chromatin samples of the Hd3a-GFP lines immunopurified using anti-GFP magnetic beads led to the identification of several novel transcription factors that bound the Hd3a FT protein and thus might form alternative transcriptionally active complex with this tuberigen signal. These included a zinc finger factor, TFs of the indeterminate (IDD) family, FRIGIDA-like, GRAS and TCP families. Surprisingly, FDL1 or ABL1 were not included in this protein dataset and therefore parallel studies were carried out with chromatin samples from stolons, to assess whether we were able to detect such bZIPs in these samples and which are currently being analyzed by MS/MS. We cloned the potato IDD, TCP, FRIGIDA-like and GRAS proteins in Y2H vectors to carry out specific protein-protein interaction studies in yeast cells to further analyze their interaction ability with the SP6A, SP5G, BRC1b, FDL1 and ABL1 baits. Our initial Y2H experiments showed very encouraging results which indicated that IDDs, TCPs, and GRAS proteins directly bind various of these tuberization regulators. We are currently verifying these results by using complementary methods like Co-IP.
As storage organs can initiate according to the plant species from different tissues, my working model in this proposal was that tuber formation relies on the vascular cambium. My initial hypothesis was that SP6A induced these meristem cells to divide, while cambium derivative cells would differentiate into storage parenchyma instead of its default pathway to xylem vessels in potato. Although, proving this mechanistic model was one of the main aims of the proposal, we could not reach to this level because of the delay in attaining the first objective, i.e. the identification of the TAC targets. As mentioned in the previous section, during last six months of Marie Curie contract I finally succeeded at identifying several SP6A targets and protein partners at the genome and proteome levels, which need further characterization.
Validation of these targets will provide fully novel mechanistic insights on the mode of action of this potato mobile protein, and specially which are its downstream regulated genes in the apical and subapical regions of the stolon. On tuberization induction the stolon shoot apical meristem arrests its growth, while cells in the subapical zone divide and expand which results in a rapid radial expansion of this zone. Although the fellowship ran to an end before I could demonstrate whether the SP6A downstream targets were specifically expressed in the apical stolon meristem or the subapical zone, I meanwhile showed through histochemical studies that stolon swelling relies on proliferation of the vascular cambium. Outcomes of this work thus not only unveiled key regulators of storage organ identity but provide a better comprehension on function of the vascular cambium hence implying a major breakthrough in our understanding on how storage plant organs are differentiated.
Validation of these targets will provide fully novel mechanistic insights on the mode of action of this potato mobile protein, and specially which are its downstream regulated genes in the apical and subapical regions of the stolon. On tuberization induction the stolon shoot apical meristem arrests its growth, while cells in the subapical zone divide and expand which results in a rapid radial expansion of this zone. Although the fellowship ran to an end before I could demonstrate whether the SP6A downstream targets were specifically expressed in the apical stolon meristem or the subapical zone, I meanwhile showed through histochemical studies that stolon swelling relies on proliferation of the vascular cambium. Outcomes of this work thus not only unveiled key regulators of storage organ identity but provide a better comprehension on function of the vascular cambium hence implying a major breakthrough in our understanding on how storage plant organs are differentiated.