BUSCADOR RECOLECTA

[EN] Microorganisms produce volatile compounds (VCs) with molecular masses of less than 300 Da that promote plant growth and photosynthesis. Recently, we have shown that small VCs of less than 45 Da other than CO2 are major determinants of plant responses to fungal volatile emissions. However, the regulatory mechanisms involved in the plants' responses to small microbial VCs remain unclear. In Arabidopsis thaliana plants exposed to small fungal VCs, growth promotion is accompanied by reduction of the thiol redox of Calvin-Benson cycle (CBC) enzymes and changes in the levels of shikimate and 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway-related compounds. We hypothesized that plants' responses to small microbial VCs involve post-translational modulation of enzymes of the MEP and shikimate pathways via mechanisms involving redox-activated photosynthesis signaling. To test this hypothesis, we compared the responses of wild-type (WT) plants and a cfbp1 mutant defective in a redox-regulated isoform of the CBC enzyme fructose-1,6-bisphosphatase to small VCs emitted by the fungal phytopathogen Alternaria alternata. Fungal VC-promoted growth and photosynthesis, as well as metabolic and proteomic changes, were substantially weaker in cfbp1 plants than in WT plants. In WT plants, but not in cfbp1 plants, small fungal VCs reduced the levels of both transcripts and proteins of the stromal Clp protease system and enhanced those of plastidial chaperonins and co-chaperonins. Consistently, small fungal VCs promoted the accumulation of putative Clp protease clients including MEP and shikimate pathway enzymes. clpr1-2 and clpc1 mutants with disrupted plastidial protein homeostasis responded weakly to small fungal VCs, strongly indicating that plant responses to microbial volatile emissions require a finely regulated plastidial protein quality control system. Our findings provide strong evidence that plant responses to fungal VCs involve chloroplast-to-nucleus retrograde signaling of redox-activated photosynthesis leading to proteostatic regulation of the MEP and shikimate pathways., This work was supported by the Agencia Estatal de Investigacion (AEI) and Fondo Europeo de Desarrollo Regional (Spain) (Grants BIO2016-78747-P, BIO2017-84041-P, BIO2017-90877-REDT, and PID2019-104685GB-100) and the Ministry of Education, Youth and Sport of the Czech Republic and ERDF project "Plants as a tool for sustainable global development" (No. CZ.02.1.01/0.0/0.0/16_019/0000827). Proteomic Unit, National Biotechnology Center was a member of Proteored, PRB3 and was supported by grant PT17/0019 of the PE I + D + i 2013-2016, funded by ISCIII and ERDF. Funding from Generalitat de Catalunya (2017SGR-710) and European Union's Horizon 2020 (EU-H2020) COST Action CA15136 (EuroCaroten) was also acknowledged. We acknowledge support of the publication fee by the CSIC Open Access Publication Support Initiative through its Unit of Information Resources for Research (URICI).

[EN] IntroductionCycling Dof transcription factors (CDFs) have been involved in different aspects of plant growth and development. In Arabidopsis and tomato, one member of this family (CDF1) has recently been associated with the regulation of primary metabolism and abiotic stress responses, but their roles in crop production under open field conditions remain unknown. MethodsIn this study, we compared the growth, and tuber yield and composition of plants ectopically expressing the CDF1 gene from Arabidopsis under the control of the 35S promoter with wild-type (WT) potato plants cultured in growth chamber and open field conditions. ResultsIn growth chambers, the 35S::AtCDF1 plants showed a greater tuber yield than the WT by increasing the biomass partition for tuber development. Under field conditions, the ectopic expression of CDF1 also promoted the sink strength of the tubers, since 35S::AtCDF1 plants exhibited significant increases in tuber size and weight resulting in higher tuber yield. A metabolomic analysis revealed that tubers of 35S::AtCDF1 plants cultured under open field conditions accumulated higher levels of glucose, starch and amino acids than WT tubers. A comparative proteomic analysis of tubers of 35S::AtCDF1 and WT plants cultured under open field conditions revealed that these changes can be accounted for changes in the expression of proteins involved in energy production and different aspects of C and N metabolism. DiscussionThe results from this study advance our collective understanding of the role of CDFs and are of great interest for the purposes of improving the yield and breeding of crop plants., We thank Manuel Sanchez-Perales and Antonio Ramos for his technical assistance and Mike Bennet for English edditing. This study was supported by the Ministerio de Ciencia e Innovacion (MCIN) and the Agencia Estatal de Investigacion (AEI)/10.13039/501100011033/(grants RTA2015-00014-c02-00 to JM and SGN; PID2020-114165RR-C21 to JM), (BIO2017-82873-R to JVC) (PID2019-104685GB-100 to JP-R, EB-F and FJM), the Gobierno de Navarra (PC036-037 BIOMEF to EB-F and FJM ) and the Vicerrectorado de Investigacion de la Universitat Politecnica de Valencia (PAID-11-21 to SGN), J.C is funded by the National Agency for Research and Development (ANID) Chile with Program FONDECYT Regular 1190812 and ANID-Millennium Science Initiative Program-ICN17_022"and The GEP Chinese pre-doctoral fellowship to LY. We also want to acknowledge the "Severo Ochoa Program for Centres of Excellence in R & D"(CEX2020-000999-S) supported by MCIN/AEI/10.13039/501100011033 for supporting the scientific services used in this study .

Introduction: cycling Dof transcription factors (CDFs) have been involved in different aspects of plant growth and development. In Arabidopsis and tomato, one member of this family (CDF1) has recently been associated with the regulation of primary metabolism and abiotic stress responses, but their roles in crop production under open field conditions remain unknown. Methods: in this study, we compared the growth, and tuber yield and composition of plants ectopically expressing the CDF1 gene from Arabidopsis under the control of the 35S promoter with wild-type (WT) potato plants cultured in growth chamber and open field conditions. Results: in growth chambers, the 35S::AtCDF1 plants showed a greater tuber yield than the WT by increasing the biomass partition for tuber development. Under field conditions, the ectopic expression of CDF1 also promoted the sink strength of the tubers, since 35S::AtCDF1 plants exhibited significant increases in tuber size and weight resulting in higher tuber yield. A metabolomic analysis revealed that tubers of 35S::AtCDF1 plants cultured under open field conditions accumulated higher levels of glucose, starch and amino acids than WT tubers. A comparative proteomic analysis of tubers of 35S::AtCDF1 and WT plants cultured under open field conditions revealed that these changes can be accounted for changes in the expression of proteins involved in energy production and different aspects of C and N metabolism. Discussion: The results from this study advance our collective understanding of the role of CDFs and are of great interest for the purposes of improving the yield and breeding of crop plants., This study was supported by the Ministerio de Ciencia e Innovación (MCIN) and the Agencia Estatal de Investigación (AEI) / 10.13039/501100011033/ (grants RTA2015-00014-c02-00 to JM and SGN; PID2020-114165RR-C21 to JM), (BIO2017-82873-R to JVC) (PID2019-104685GB-100 to JP-R, EB-F and FJM), the Gobierno de Navarra (PC036-037 BIOMEF to EB-F and FJM) and the Vicerrectorado de Investigación de la Universitat Politècnica de València (PAID-11-21 to SGN), J.C is funded by the National Agency for Research and Development (ANID) Chile with Program FONDECYT Regular 1190812 and ANID-Millennium Science Initiative Program-ICN17_022" and The GEP Chinese pre-doctoral fellowship to LY. We also want to acknowledge the "Severo Ochoa Program for Centres of Excellence in R&D" (CEX2020-000999-S) supported by MCIN/AEI/10.13039/501100011033 for supporting the scientific services used in this study.

Plants communicate with microorganisms by exchanging chemical signals throughout the phytosphere. Such interactions are important not only for plant productivity and fitness, but also for terrestrial ecosystem functioning. It is known that beneficial microorganisms emit diffusible substances including volatile organic compounds (VOCs) that promote growth. Consistently, soil application of cell-free culture filtrates (CF) of beneficial soil and plant-associated microorganisms enhances plant growth and yield. However, how this treatment acts in plants and whether it alters the resident soil microbiota, are largely unknown. In this work we characterized the responses of pepper (Capsicum annuum L.) plants cultured under both greenhouse and open field conditions and of soil microbiota to soil application of CFs of beneficial and phytopathogenic fungi. To evaluate the contribution of VOCs occurring in the CFs to these responses, we characterized the responses of plants and of soil microbiota to application of distillates (DE) of the fungal CFs. CFs and their respective DEs contained the same potentially biogenic VOCs, and application of these extracts enhanced root growth and fruit yield, and altered the nutritional characteristics of fruits. High-throughput amplicon sequencing of bacterial 16S and fungal ITS rRNA genes of the soil microbiota revealed that the CF and DE treatments altered the microbial community compositions, and led to strong enrichment of the populations of the same beneficial bacterial and fungal taxa. Our findings show that CFs of both beneficial and phytopathogenic fungi can be used as biostimulants, and provide evidence that VOCs occurring in the fungal CFs act as mediators of the plants’ responses to soil application of fungal CFs through stimulation of the beneficial soil microbiota., This work was supported by the Agencia Estatal de Investigación (AEI) and Fondo Europeo de Desarrollo Regional (Spain) (grants BIO2013-49125-C2-1-P, BIO2016-78747-P, and PID2019-104685GB-100), the Government of Navarra (refs. P1044 AGROESTI, P1004 PROMEBIO, and P1046 MICROBIOME), and the project “Plants as a tool for sustainable global development” (registration number: CZ.02.1.01/0.0/0.0/16_019/0000827) within the program Research, Development and Education (OP RDE).

In this work we compiled information on current and emerging microbial-based fertilization practices, especially the use of cell-free microbial culture filtrates (CFs), to promote plant growth, yield and stress tolerance, and their effects on plant-associated beneficial microbiota. In addition, we identified limitations to bring microbial CFs to the market as biostimulants. In nature, plants act as metaorganisms, hosting microorganisms that communicate with the plants by exchanging semiochemicals through the phytosphere. Such symbiotic interactions are of high importance not only for plant yield and quality, but also for functioning of the soil microbiota. One environmentally sustainable practice to increasing crop productivity and/or protecting plants from (a)biotic stresses while reducing the excessive and inappropriate application of agrochemicals is based on the use of inoculants of beneficial microorganisms. However, this technology has a number of limitations, including inconsistencies in the field, specific growth requirements and host compatibility. Beneficial microorganisms release diffusible substances that promote plant growth and enhance yield and stress tolerance. Recently, evidence has been provided that this capacity also extends to phytopathogens. Consistently, soil application of microbial cell-free culture filtrates (CFs) has been found to promote growth and enhance the yield of horticultural crops. Recent studies have shown that the response of plants to soil application of microbial CFs is associated with strong proliferation of the resident beneficial soil microbiota. Therefore, the use of microbial CFs to enhance both crop yield and stress tolerance, and to activate beneficial soil microbiota could be a safe, efficient and environmentally friendly approach to minimize shortfalls related to the technology of microbial inoculation. In this review, we compile information on microbial CFs and the main constituents (especially volatile compounds) that promote plant growth, yield and stress tolerance, and their effects on plant-associated beneficial microbiota. In addition, we identify challenges and limitations for their use as biostimulants to bring them to the market and we propose remedial actions and give suggestions for future work., This work was supported by the Agencia Estatal de Investigación (AEI) and Fondo Europeo de Desarrollo Regional (Spain) (grant PID2019-104685GB-100).

Resumen del trabajo presentado en la XXIV Reunión de la Sociedad Española de Biología de Plantas - XVII Congreso Hispano-Luso de Biología de Plantas, celebrado online del 7 al 9 de julio de 2021, Recent studies have shown that PGI1 is an important determinant of
photosynthesis, growth and starch production, likely as a consequence of its involvement in
the synthesis of plastidial isoprenoid compounds such as hormones and photosynthetic
pigments (Bahaji et al. 2015; Bahaji et al. 2018). We have shown that volatile compounds
(VCs) emitted by microbes promote growth, photosynthesis, and accumulation of cytokinins
(CK) and starch (Sánchez-Lopez et al. 2016a). This response was PGI1-independent, as
fungal VCs exposed PGI1 null pgi1-2 plants grew faster and accumulated exceedingly
higher levels of starch and CKs than plants not exposed to VCs (Sánchez-López et al.
2016b). The most up-regulated gene in leaves of fungal VCs exposed plants was At1g61800,
encoding the plastidial GPT2 glucose-6-phosphate (G6P)/phosphate translocator. Whether
the response of pgi1-2 to fungal VCs could be due to the GPT2-mediated transport of
cytosolic G6P into the chloroplast was investigated by characterizing pgi1-2/gpt2-2 PGI1-
and GPT2-null double mutants cultured in the presence or absence of VCs emitted by the
fungal phytopathogen Alternaria alternata. We found that photosynthesis, active CKs
content, growth and leaf starch content in pgi1-2/gpt2-2 plants exposed to fungal VCs were
lower than in VCs exposed WT, gpt2-2 and pgi1-2 plants. Proteomic analyses revealed that
fungal VCs strongly up-regulate the expression of proteins involved in photosynthesis in
WT, gpt2-2 and pgi1-2 plants, but in much lower extent in pgi1-2/gpt2-2 plants. The overall data show that the combined action of PGI1 and GTP2 is an important determinant of the
plant´s response to microbial VCs. The possible involvement of these functions in the
production of growth- and foliar metabolism-regulating isoprenoid hormones in
heterotrophic organs is discussed., This work was supported by the
Agencia Estatal de Investigación (AEI) and Fondo Europeo de Desarrollo Regional (Spain)
(grants BIO2016-78747-P and PID2019-104685GB-100) and the Ministry of Education,
Youth and Sport of the Czech Republic and ERDF project “Plants as a tool for sustainable
global development” (No. CZ.02.1.01/0.0/0.0/16_019/0000827)., Peer reviewed

Resumen del trabajo presentado en la XXIV Reunión de la Sociedad Española de Biología de Plantas - XVII Congreso Hispano-Luso de Biología de Plantas, celebrado online del 7 al 9 de julio de 2021, Microbes synthesize a multitude of substances including carbohydrates,
proteins, lipids, amino acids, hormones, volatiles, etc., which regulate plant growth and
morphogenesis. Irrigation with culture filtrates (CFs) of some plant beneficial
microorganisms promotes plant growth and enhances yield. However, nothing is known
about the response of plants to soil application of CFs of microbial phytopathogens. In this
work we explored the response of two pepper (Capsicum annuum L.) cultivars (i.e. Sweet
Italian and Piquillo) and soil microbiota to CFs of the beneficial fungus (i.e. Trichoderma
harzianum) and two fungal phytopathogens (i.e. Alternaria alternata and Penicilliun
aurantiogriseum). We found that soil application of CFs of the three fungal species
similarly enhanced root growth (40-70% increase under greenhouse conditions) and
commercial fruit yield (50-112% and 20-28% increase under greenhouse and open field
conditions, respectively). High-throughput amplicon sequencing of bacterial 16S and
fungal ITS rRNA genes of the soil microbiota revealed that the CF treatments led to strong
enrichment of the populations of the same beneficial bacterial and fungal species. Our
findings show that the application of microbial CFs to the soil represents a potentially very
useful strategy for increasing the yield of horticultural crops in a sustainable and
environmentally friendly way, This work was supported by the Agencia Estatal de Investigación
(AEI) and Fondo Europeo de Desarrollo Regional (Spain) (grants BIO2013-49125-C2-1-
P, BIO2016-78747-P and PID2019-104685GB-100), the Government of Navarra (refs.
P1044 AGROESTI, P1004 PROMEBIO and P1046 MICROBIOME), and the Ministry of Education, Youth and Sport of the Czech Republic and the ERDF project “Plants as a tool
for sustainable global development” (No. CZ.02.1.01/0.0/0.0/16_019/0000827).

In Arabidopsis (Arabidopsis thaliana), the plastidial isoform of phosphoglucose isomerase (PGI1) mediates photosynthesis, metabolism, and development, probably due to its involvement in the synthesis of isoprenoid-derived signals in vascular tissues. Microbial volatile compounds (VCs) with molecular masses of <45 Da promote photosynthesis, growth, and starch overaccumulation in leaves through PGI1-independent mechanisms. Exposure to these compounds in leaves enhances the levels of GLUCOSE-6-PHOSPHATE/PHOSPHATE TRANSLOCATOR2 (GPT2) transcripts. We hypothesized that the PGI1-independent response to microbial volatile emissions involves GPT2 action. To test this hypothesis, we characterized the responses of wild-type (WT), GPT2-null gpt2-1, PGI1-null pgi1-2, and pgi1-2gpt2-1 plants to small fungal VCs. In addition, we characterized the responses of pgi1-2gpt2-1 plants expressing GPT2 under the control of a vascular tissue- and root tip-specific promoter to small fungal VCs. Fungal VCs promoted increases in growth, starch content, and photosynthesis in WT and gpt2-1 plants. These changes were substantially weaker in VC-exposed pgi1-2gpt2-1 plants but reverted to WT levels with vascular and root tip-specific GPT2 expression. Proteomic analyses did not detect enhanced levels of GPT2 protein in VC-exposed leaves and showed that knocking out GPT2 reduced the expression of photosynthesis-related proteins in pgi1-2 plants. Histochemical analyses of GUS activity in plants expressing GPT2-GUS under the control of the GPT2 promoter showed that GPT2 is mainly expressed in root tips and vascular tissues around hydathodes. Overall, the data indicated that the PGI1-independent response to microbial VCs involves resetting of the photosynthesis-related proteome in leaves through long-distance GPT2 action., This work was supported by the Ministerio de Ciencia e Innovación (MCIN) and Agencia Estatal de Investigación (AEI)/10.13039/501100011033/ (grants BIO2016-78747-P, PID2019-104685GB-100 and PID2019-107657RB-C22) and the Ministry of Education, Youth and Sport of the Czech Republic and the European Regional Development Fund (ERDF) project entitled “Plants as a tool for sustainable global development” (No. CZ.02.1.01/0.0/0.0/16_019/0000827)., Peer reviewed

Resumen del trabajo presentado en el International Symposium: Microbe-Assisted Crop Production - Opportunities, Challenges & Needs (MICROPE 2022), celebrado en Viena (Austria), del 11 al 14 de julio de 2022, Microorganisms emit a plethora of volatile compounds (VCs) that promote plant growth and photosynthesis as well as strong developmental and metabolic changes. Recently, we showed that small VCs of molecular masses of less than ca. 45 Da are important determinants of plant responses to microbial volatile emissions. In Arabidopsis, the plastidial isoform of phosphoglucose isomerase PGI1 mediates photosynthesis, metabolism and development, probably due to its involvement in the synthesis of isoprenoid-derived signals in vascular tissues. Like in wild-type (WT) plants, small VCs promote growth and photosynthesis as well as starch and CK accumulation in PGI1-lacking pgi1-2 plants. A striking alteration in the transcriptome of leaves of small fungal VC-treated plants involves strong up-regulation of levels of transcripts of GPT2 (At1g61800), a gene that codes for a plastidial G6P/Pi transporter. We hypothesized that the PGI1-independent response to microbial volatile emissions involves GPT2 action. To test this hypothesis, we characterized responses of WT, GPT2-null gpt2-2, PGI1-null pgi1-2 and pgi1-2gpt2-2 plants to small fungal VCs. In addition, we characterized responses of pgi1-2gpt2-2 plants expressing GPT2 under the control of a vascular tissue- and root tip-specific promoter to small fungal VCs. We found that the small VC-promoted changes in pgi1-2gpt2-2 plants were substantially weaker than in WT, gpt2-2 and pgi1-2 plants but reverted to WT levels by vascular and root tip-specific GPT2 expression. Proteomic analyses did not detect enhanced levels of GPT2 protein in VC-exposed leaves. Results presented in this work provide evidence that, under conditions in which PGI1 activity is reduced, long-distance action of GPT2 plays an important role in the response of plants to small VCs through mechanisms involving resetting of the photosynthesis-related proteome in leaves and complex GPT2 regulation., This work was supported by the Ministerio de Ciencia e Innovación (MCIN) and Agencia Estatal de Investigación (AEI) / 10.13039/501100011033/ (grants BIO2016-78747-P, PID2019-104685GB-100 and PID2019-107657RB-C22) and the Ministry of Education, Youth and Sport of the Czech Republic and ERDF project entitled “Plants as a tool for sustainable global development” (No. CZ.02.1.01/0.0/0.0/16_019/0000827)

Resumen del trabajo presentado en el International Symposium Microbe-assisted crop production – opportunities, challenges and needs (MICROPE 2022), celebrado en Viena (Austria), del 11 al 14 de julio de 2022, Plants communicate with microorganisms by exchanging chemical signals throughout the phytosphere. Such interactions are important not only for plant productivity and fitness, but also for terrestrial ecosystem functioning. It is known that beneficial microorganisms emit diffusible substances including volatile organic compounds (VOCs) that promote growth. Consistently, soil application of cell-free culture filtrates (CF) of beneficial soil and plant-associated microorganisms enhances plant growth and yield. However, how this treatment acts in plants and whether it alters the resident soil microbiota, are largely unknown. In this work we characterized the responses of pepper (Capsicum annuum L.) plants cultured under both greenhouse and open field conditions and of soil microbiota to soil application of CFs of beneficial and phytopathogenic fungi. To evaluate the contribution of VOCs occurring in the CFs to these responses, we characterized the responses of plants and of soil microbiota to application of distillates (DE) of the fungal CFs. CFs and their respective DEs contained the same potentially biogenic VOCs, and application of these extracts enhanced root growth and fruit yield, and altered the nutritional characteristics of fruits. High-throughput amplicon sequencing of bacterial 16S and fungal ITS rRNA genes of the soil microbiota revealed that the CF and DE treatments altered the microbial community compositions, and led to strong enrichment of the populations of the same beneficial bacterial and fungal taxa. Our findings show that CFs of both beneficial and phytopathogenic fungi can be used as biostimulants, and provide evidence that VOCs occurring in the fungal CFs act as mediators of the plants’ responses to soil application of fungal CFs through stimulation of the beneficial soil microbiota., This work was supported by the Agencia Estatal de Investigación (AEI) and Fondo Europeo de Desarrollo Regional (Spain) (grants BIO2013-49125-C2-1-P, BIO2016-78747-P and PID2019-104685GB-100), the Government of Navarra (refs. P1044 AGROESTI, P1004 PROMEBIO and P1046 MICROBIOME), and the project “Plants as a tool for sustainable global development” (registration number: CZ.02.1.01/0.0/0.0/16_019/0000827) within the program Research, Development and Education (OP RDE)

Resumen del trabajo presentado en el XVI Meeting of Plant Molecular Biology, celebrado en Sevilla (España), del 14 al 16 de septiembre de 2022, Phosphoglucose isomerase is involved in the early steps of glycolysis and regeneration of
glucose-6-phosphate pools in the pentose phosphate pathway (PPP). In Arabidopsis, plastidial
phosphoglucose isomerase (PGI1) is an important determinant of growth, metabolism and
photosynthesis, probably due to its involvement in the synthesis of 2-C-methyl-D-erythritol 4-P
(MEP)-derived hormones in root tips and vascular tissues (Bahaji et al., 2015; Bahaji et al., 2018).
To test this hypothesis, we conducted proteomic and metabolic characterization of PGI1-null
pgi1-2 plants. We also characterized pgi1-2 plants ectopically expressing PGI1 under the control
of a root tip- and vascular tissue-specific promoter. Furthermore, we characterized pfk4/pfk5
knockout plants impaired in the early steps of plastidial glycolysis, and pgl3-1 plants with reduced
activity of the plastidial PPP enzyme 6-phosphogluconolactonase 3. The overall data obtained
in this work provide strong evidence that root tip and vascular PGI1-mediated plastidial PPP
determines growth, development and photosynthesis through MEP pathway action., This work was supported by the Ministerio de Ciencia e Innovación (MCIN) and Agencia Estatal de Investigación (AEI)
/ 10.13039/501100011033/ (grants BIO2016-78747-P, PID2019-104685GB-100) and the Ministry of Education, Youth
and Sport of the Czech Republic and ERDF project entitled “Plants as a tool for sustainable global development” (No.
CZ.02.1.01/0.0/0.0/16_019/0000827).

Resumen del trabajo presentado en el XVI Meeting of Plant Molecular Biology, celebrado en Sevilla (España), del 14 al 16 de septiembre de 2022, Microorganisms emit a plethora of volatile compounds (VCs) that promote plant growth and
photosynthesis as well as strong developmental and metabolic changes. In Arabidopsis, the
plastidial isoform of phosphoglucose isomerase PGI1 mediates photosynthesis, metabolism and
development, probably due to its involvement in the synthesis of isoprenoid-derived signals in
vascular tissues (Bahaji et al., 2015; Bahaji et al., 2018). Like in wild-type (WT) plants, microbial
VCs promote growth and photosynthesis as well as starch and CK accumulation in PGI1-lacking
pgi1-2 plants (Sánchez-López et al. 2016). A striking alteration in the transcriptome of leaves
of small fungal VC-treated plants involves strong up-regulation of levels of transcripts of GPT2
(At1g61800), a gene that codes for a plastidial G6P/Pi transporter. We hypothesized that the
PGI1-independent response to microbial volatile emissions involves GPT2 action. To test this
hypothesis, we characterized responses of WT, GPT2-null gpt2-1, PGI1-null pgi1-2 and pgi1-
2gpt2-1 plants to small fungal VCs. In addition, we characterized responses of pgi1-2gpt2-1
plants expressing GPT2 under the control of a vascular tissue- and root tip-specific promoter to
small fungal VCs. Results presented in this work provide evidence that, under conditions in which
PGI1 activity is reduced, long-distance action of GPT2 plays an important role in the response
of plants to small VCs through mechanisms involving resetting of the photosynthesis-related
proteome in leaves and complex GPT2 regulation., This work was supported by the Ministerio de Ciencia e Innovación (MCIN) and Agencia Estatal de Investigación (AEI)
/ 10.13039/501100011033/ (grants BIO2016-78747-P, PID2019-104685GB-100 and PID2019-107657RB-C22) and the
Ministry of Education, Youth and Sport of the Czech Republic and ERDF project entitled “Plants as a tool for sustainable
global development” (No. CZ.02.1.01/0.0/0.0/16_019/0000827).

In this work we compiled information on current and emerging microbial-based fertilization practices, especially the use of cell-free microbial culture filtrates (CFs), to promote plant growth, yield and stress tolerance, and their effects on plant-associated beneficial microbiota. In addition, we identified limitations to bring microbial CFs to the market as biostimulants. In nature, plants act as metaorganisms, hosting microorganisms that communicate with the plants by exchanging semiochemicals through the phytosphere. Such symbiotic interactions are of high importance not only for plant yield and quality, but also for functioning of the soil microbiota. One environmentally sustainable practice to increasing crop productivity and/or protecting plants from (a)biotic stresses while reducing the excessive and inappropriate application of agrochemicals is based on the use of inoculants of beneficial microorganisms. However, this technology has a number of limitations, including inconsistencies in the field, specific growth requirements and host compatibility. Beneficial microorganisms release diffusible substances that promote plant growth and enhance yield and stress tolerance. Recently, evidence has been provided that this capacity also extends to phytopathogens. Consistently, soil application of microbial cell-free culture filtrates (CFs) has been found to promote growth and enhance the yield of horticultural crops. Recent studies have shown that the response of plants to soil application of microbial CFs is associated with strong proliferation of the resident beneficial soil microbiota. Therefore, the use of microbial CFs to enhance both crop yield and stress tolerance, and to activate beneficial soil microbiota could be a safe, efficient and environmentally friendly approach to minimize shortfalls related to the technology of microbial inoculation. In this review, we compile information on microbial CFs and the main constituents (especially volatile compounds) that promote plant growth, yield and stress tolerance, and their effects on plant-associated beneficial microbiota. In addition, we identify challenges and limitations for their use as biostimulants to bring them to the market and we propose remedial actions and give suggestions for future work, This work was supported by the Agencia Estatal de Investigación (AEI) and Fondo Europeo de Desarrollo Regional (Spain) (grant PID2019-104685GB-100).

Resumen del trabajo presentado en el XVI Meeting Of Plant Molecular Biology (XVI RBMP), celebrado en Sevilla (España), del 14 al 16 de septiembre de 2022, Microorganisms emit volatile compounds (VCs) that promote plant growth and photosynthesis as
well as strong developmental and metabolic changes through multiple and complex regulatory
mechanisms including global reduction of the thiol redox proteome (Gámez-Arcas et al. 2022).
Recently, we found that microbial VC treatment promotes the reduction of the Cys₁₅₄ residue of the
redox-regulated Calvin-Benson cycle enzyme fructose-1,6-bisphosphatase 1 (cFBP1) (Ameztoy et
al. 2019). Although highly conserved throughout land plants and algae, this residue is not located
in the proposed regulatory Trx redox or catalytic domains of cFBP1. To investigate the role played
by Cys¹⁵⁴ in the activity of cFBP1 and the response of plants to microbial VCs, we produced and
characterized recombinantly produced wild type (WT) cFBP1 (cFBP1wt) and a mutated form of
cFBP1 in which the Cys₁₅₄ residue has been replaced by serine (cFBP1mut). We also produced
and characterized cfbp1 plants ectopically expressing cFBP1wt and cFBP1mut under the control
of the cFBP1 promoter. In native gels, the electrophoretic mobilities of recombinantly produced
cFBP1 and cFBP1mut were different. In addition, recombinant cFBP1mut had ca. 85% less activity
than cFBP1wt. The ectopic expression of cFBP1wt, and to a lesser extent that of cFBP1mut,
countered the reduced photosynthetic activity of cFBP1-lacking cfbp1 plants, reverting it to the
WT. Results presented in this work provide strong evidence that the Cys₁₅₄ residue of cFBP1 is an
important determinant of photosynthetic activity in Arabidopsis., This work was supported by the Ministerio de Ciencia e Innovación (MCIN) and Agencia Estatal de Investigación (AEI) /
10.13039/501100011033/ (grants BIO2016-78747-P, PID2019-104685GB-100 and PGC2018-096851-B-C21)., Peer reviewed

16 Pág., Cycling Dof transcription factors (CDFs) have been involved in different aspects of plant growth and development. In Arabidopsis and tomato, one member of this family (CDF1) has recently been associated with the regulation of primary metabolism and abiotic stress responses, but their roles in crop production under open field conditions remain unknown., This study was supported by the Ministerio de Ciencia e Innovación (MCIN) and the Agencia Estatal de Investigación (AEI) / 10.13039/501100011033/ (grants RTA2015-00014-c02-00 to JM and SGN; PID2020-114165RR-C21 to JM), (BIO2017-82873-R to JVC) (PID2019-104685GB-100 to JP-R, EB-F and FJM), the Gobierno de Navarra (PC036-037 BIOMEF to EB-F and FJM) and the Vicerrectorado de Investigación de la Universitat Politècnica de València (PAID-11-21 to SGN), J.C is funded by the National Agency for Research and Development (ANID) Chile with Program FONDECYT Regular 1190812 and ANID-Millennium Science Initiative Program-ICN17_022" and The GEP Chinese pre-doctoral fellowship to LY. We also want to acknowledge the "Severo Ochoa Program for Centres of Excellence in R&D" (CEX2020-000999-S) supported by MCIN/AEI/10.13039/501100011033 for supporting the scientific services used in this study., With funding from the Spanish government through the ‘Severo Ochoa Centre of Excellence’ accreditation (CEX2020‐000999‐S), Peer reviewed

Tesis doctoral presentada para lograr el título de Doctor por la Universidad Pública de Navarra, Departamento de Agronomía, Biotecnología y Alimentación, Programa de Doctorado en Biotecnología, Las plantas son metaorganismos que regulan su desarrollo y metabolismo en respuesta a diferentes estímulos externos. Conviven con microorganismos emisores de compuestos que difunden a través del suelo tales como azúcares, proteínas, exopolisacáridos, sideróforos, ácidos grasos, ácidos orgánicos, aminoácidos y hormonas que modifican la fotosíntesis, el metabolismo y el crecimiento de las plantas (Philippot et al., 2013; De-la-Peña and Loyola-Vargas, 2014; Backer et al., 2018). Los microorganismos beneficiosos también emiten compuestos volátiles (VCs) que fomentan el crecimiento, la captación de nutrientes y la fotosíntesis, modulan la arquitectura de la raíz, activan la respuesta de defensa de la planta e incrementan su resistencia al estrés salino y la sequía (Ryu et al., 2003; Zhang et al., 2008; Splivallo et al., 2009; Zhang et al., 2009; Gutiérrez-Luna et al., 2010; Kanchiswamy et al., 2015; Park et al., 2015; Ledger et al., 2016). Estudios llevados a cabo por el grupo de investigación en el que he realizado mi tesis doctoral demostraron que las bacterias y los hongos fitopatógenos también emiten VCs con propiedades bioestimulantes (Sánchez-López et al., 2016b). Cambios en el crecimiento, la fotosíntesis, el desarrollo y el metabolismo de la planta inducidos por VCs microbianos están asociados a grandes variaciones en el transcriptoma, el proteoma y el redox-proteoma de la planta (Sánchez- López et al., 2016b; Ameztoy et al., 2019, 2021). Tales variaciones sugieren que la respuesta de la planta a los VCs microbianos está regulada tanto a nivel transcripcional como no-transcripcional. Con el fin de profundizar en los mecanismos que median la relación entre la planta y los microorganismos, en este trabajo investigué el papel que juega el transportador de hexosas fosfato plastidial (GPT2) inducible por VCs en la respuesta de la planta modelo de laboratorio Arabidopsis thaliana a VCs emitidos por el hongo fitopatógeno Altenaria alternata. Además, investigué el papel que juega en dicha respuesta el residuo de cisteína 95 de la fructosa bifosfatasa plastidial (cFBP1), un enzima del ciclo de Calvin-Benson cuya actividad está sujeta a cambios en el estado redox inducibles por la luz. En el capítulo 1 de esta tesis presento los resultados de mi investigación sobre el papel que juega GPT2 en la respuesta de las plantas a VCs microbianos. Para realizar esta investigación, analicé y comparé el crecimiento, la fotosíntesis y el proteoma de plantas wild type (WT), mutantes carentes GPT2 (gpt2-1), mutantes carentes PGI1 (pgi1-2) y doble mutante pgi1-2gpt2-1 cultivadas en presencia o ausencia de VCs fúngicos. Además, caractericé la respuesta a estos compuestos de plantas pgi1-2/gpt2-1 que expresan GPT2 bajo el control de un promotor específico de haces vasculares. Finalmente, caractericé el patrón de expresión de GPT2 en plantas que expresan el gen reportero GUS bajo la acción de la secuencia promotora de GPT2. Los resultados obtenidos mostraron que, de manera similar a lo que ocurre en plantas WT, los VCs fomentan el crecimiento, la fotosíntesis y la acumulación de citoquininas activas y almidón en plantas pgi1-2 y gpt2-1. Tal respuesta fue reducida en plantas pgi1-2gpt2-1 y se revirtió a WT al expresar GPT2 en haces vasculares., Estudios comparativos de los proteomas de plantas pgi1-2 y pgi1-2gpt2-1 mostraron que GPT2 regula la expresión de proteínas relacionadas con la fotosíntesis. Es más, los estudios de expresión mostraron que GPT2 se expresa en haces vasculares y está sujeto a complejos mecanismos de regulación. Globalmente, los resultados presentados en este capítulo indican que en condiciones en las cuales la actividad de PGI está reducida, la respuesta de la planta a VCs microbianos conlleva una regulación de la expresión de proteínas relacionadas con la fotosíntesis a través de mecanismos en los que GPT2 juega un papel importante en la provisión de sustrato necesario para la producción de citoquininas en haces vasculares. Trabajos llevados a cabo en el grupo de investigación en el que he realizado este mi tesis mostraron que la exposición a VCs microbianos conlleva cambios globales en el redox-proteoma de las plantas (Ameztoy et al., 2019). Tales cambios incluían la reducción de residuos de cisteína altamente conservados a lo largo de la evolución de enzimas del ciclo de Calvin-Benson. En el capítulo 2 presento los resultados de mi investigación sobre el papel que juega el residuo de cisteína 95 de la cFBP1 en su actividad, en la fotosíntesis y en la respuesta de las plantas a VCs microbianos. Para realizar esta investigación, comparé las propiedades electroforéticas y cinéticas de cFBP1 WT y una forma mutada en la que el residuo de cisteína 95 fue sustituido por un residuo de serina (C95S). Además, comparé la fotosíntesis y el crecimiento de plantas de Arabidopsis cfbp1 carentes de cFBP1 endógena que expresan ectópicamente formas WT o C95S de cFBP1. Los resultados mostraron que el residuo de cisteína 95 juega un papel importante en la actividad de cFBP1 y en la fotosíntesis, pero no en el crecimiento de Arabidopsis., Beca predoctoral de la Universidad Pública de Navarra; Ministerio de Ciencia e Innovación (MICINN) y Agencia Estatal de Investigación (AEI)/10.13039/501100011033/(proyectos BIO2016-78747-P y PID2019-104685GB-100).

1 página - Poster presentado en Iberian Plant Biology 2023. XVIII Portuguese-Spanish Congress on Plant Biology and the XXV Meeting of the Spanish Society of Plant Biology. 9-12 Julio 2023, Braga, Portugal., This work was supported by the Ministerio de Ciencia e Innovación (MCIN) and Agencia Estatal de Investigación (AEI) / 10.13039/501100011033/ (grants BIO2016‐78747‐P, PID2019‐104685GB‐100 and PGC2018‐096851‐B‐C21).

RAPID ALKALINIZATION FACTOR (RALF) peptides are important players in regulating cell expansion. In Arabidopsis, volatile compounds (VCs) emitted by the fungal phytopathogen Penicillium aurantiogriseum promote root hair (RH) proliferation and hyper-elongation through ethylene and enhanced photosynthesis signalling actions. A striking alteration in the proteome of fungal VC-treated roots involves up-regulation of RALF22. To test the possible involvement of RALF22 in the fungal VC-promoted RH changes, we characterized RH density and number responses to fungal VCs in ralf22 and fer-4 plants impaired in RALF22 and its receptor FERONIA, respectively. Unlike WT plants, ralf22 and fer-4 RHs responded weakly to fungal VCs, strongly indicating that the RALF22-FERONIA module is a key determinant of the RH response to fungal VCs. To investigate the regulatory mechanisms behind this response, we analysed the RALF22 transcript levels in roots of etr1-3 and eir1 ethylene signalling mutants and those of ethylene-responsive, RH-related RSL4, RHD2, PRX1 and PRX44 transcripts in ralf22 and fer-4 roots. Moreover, we characterized the RH and RALF22 transcript level responses to fungal VCs of the cfbp1 mutant defective in photosynthetic responsiveness to VCs. Unlike in WT roots, fungal VCs weakly enhanced RALF22 expression in etr1-3, eir1 and cfbp1 roots, and RSL4, RHD2, PRX1 and PRX44 expression in ralf22 and fer-4 roots. In addition, fungal VCs weakly promoted RH changes in cfbp1 roots. Collectively, our findings showed that the ethylene and enhanced photosynthesis signalling-mediated RH response to fungal VCs involves RALF22-FERONIA., This work was supported by the Ministerio de Ciencia e Innovación (MCIN) and Agencia
Estatal de Investigación (AEI) / 10.13039/501100011033/ (grant PID2019-104685GB-100).

In Arabidopsis, the plastidial isoform of phosphoglucose isomerase, PGI1, mediates growth and photosynthesis, likely due to its involvement in the vascular production of cytokinins (CK). To examine this hypothesis, we characterized pgi1-2 knockout plants impaired in PGI1 and pgi1-2 plants specifically expressing PGI1 in root tips and vascular tissues. Moreover, to investigate whether the phenotype of pgi1-2 plants is due to impairments in the plastidial oxidative pentose phosphate pathway (OPPP) or the glycolytic pathway, we characterized pgl3-1 plants with reduced OPPP and pfk4pfk5 knockout plants impaired in plastidial glycolysis. Compared with wild-type (WT) leaves, pgi1-2 leaves exhibited weaker expression of photosynthesis- and 2-C-methyl-D-erythritol 4-P (MEP) pathway-related proteins, and stronger expression of oxidative stress protection-related enzymes. Consistently, pgi1-2 leaves accumulated lower levels of chlorophyll, and higher levels of tocopherols, flavonols and anthocyanins than the WT. Vascular- and root tip-specific PGI1 expression countered the reduced photosynthesis, low MEP pathway-derived CK content, dwarf phenotype and the metabolic characteristics of pgi1-2 plants, reverting them to WT-like levels. Moreover, pgl3-1, but not pfk4pfk5 plants phenocopied pgi1-2. Histochemical analyses of plants expressing GUS under the control of promoter regions of genes encoding plastidial OPPP enzymes exhibited strong GUS activity in root tips and vascular tissues. Overall, our findings show that root tip and vascular PGI1-mediated plastidial OPPP activity affects photosynthesis and growth through mechanisms involving long-distance modulation of the leaf proteome by MEP pathway-derived CKs., This work was supported by the Ministerio de Ciencia e Innovación (MCIN) and Agencia Estatal de Investigación (AEI)/10.13039/501100011033/(grants BIO2016-78747-P, PID2019-104685GB-100) and the Ministry of Education, Youth and Sport of the Czech Republic (No. CZ.02.1.01/0.0/0.0/16_019/0000827) within the program Research, Development, and Education (OP RDE)., Peer reviewed