Perturbation of protein homeostasis brings plastids at the crossroad between repair and dismantling.

The chloroplast proteome is a dynamic mosaic of plastid- and nuclear-encoded proteins. Plastid protein homeostasis is maintained through the balance between de novo synthesis and proteolysis. Intracellular communication pathways, including the plastid-to-nucleus signalling and the protein homeostasis machinery, made of stromal chaperones and proteases, shape chloroplast proteome based on developmental and physiological needs. However, the maintenance of fully functional chloroplasts is costly and under specific stress conditions the degradation of damaged chloroplasts is essential to the maintenance of a healthy population of photosynthesising organelles while promoting nutrient redistribution to sink tissues. In this work, we have addressed this complex regulatory chloroplast-quality-control pathway by modulating the expression of two nuclear genes encoding plastid ribosomal proteins PRPS1 and PRPL4. By transcriptomics, proteomics and transmission electron microscopy analyses, we show that the increased expression of PRPS1 gene leads to chloroplast degradation and early flowering, as an escape strategy from stress. On the contrary, the overaccumulation of PRPL4 protein is kept under control by increasing the amount of plastid chaperones and components of the unfolded protein response (cpUPR) regulatory mechanism. This study advances our understanding of molecular mechanisms underlying chloroplast retrograde communication and provides new insights into cellular responses to impaired plastid protein homeostasis.

The cyclic peptide G4CP2 enables the modulation of galactose metabolism in yeast by interfering with GAL4 transcriptional activity.

Genetically-encoded combinatorial peptide libraries are convenient tools to identify peptides to be used as therapeutics, antimicrobials and functional synthetic biology modules. Here, we report the identification and characterization of a cyclic peptide, G4CP2, that interferes with the GAL4 protein, a transcription factor responsible for the activation of galactose catabolism in yeast and widely exploited in molecular biology. G4CP2 was identified by screening CYCLIC, a Yeast Two-Hybrid-based combinatorial library of cyclic peptides developed in our laboratory. G4CP2 interferes with GAL4-mediated activation of galactose metabolic enzymes both when expressed intracellularly, as a recombinant peptide, and when provided exogenously, as a chemically-synthesized cyclic peptide. Our results support the application of G4CP2 in microbial biotechnology and, additionally, demonstrate that CYCLIC can be used as a tool for the rapid identification of peptides, virtually without any limitations with respect to the target protein. The possible biotechnological applications of cyclic peptides are also discussed.

GUN1 involvement in the redox changes occurring during biogenic retrograde signaling.

Chloroplast biogenesis requires a tight communication between nucleus and plastids. By retrograde signals, plastids transmit information about their functional and developmental state to adjust nuclear gene expression, accordingly. GENOMES UNCOUPLED 1 (GUN1), a chloroplast-localized protein integrating several developmental and stress-related signals, is one of the main players of retrograde signaling. Here, we focused on the interplay between GUN1 and redox regulation during biogenic retrograde signaling, by investigating redox parameters in Arabidopsis wild type and gun1 seedlings. Our data highlight that during biogenic retrograde signaling superoxide anion (O2-) and hydrogen peroxide (H2O2) play a different role in response to GUN1. Under physiological conditions, even in the absence of a visible phenotype, gun1 mutants show low activity of superoxide dismutase (SOD) and ascorbate peroxidase (APX), with an increase in O2- accumulation and lipid peroxidation, suggesting that GUN1 indirectly protects chloroplasts from oxidative damage. In wild type seedlings, perturbation of chloroplast development with lincomycin causes H2O2 accumulation, in parallel with the decrease of ROS-removal metabolites and enzymes. These redox changes do not take place in gun1 mutants which, in contrast, enhance SOD, APX and catalase activities. Our results indicate that in response to lincomycin, GUN1 is necessary for the H2O2-dependent oxidation of cellular environment, which might contribute to the redox-dependent plastid-to nucleus communication.

Chloroplast-localized GUN1 contributes to the acquisition of basal thermotolerance in Arabidopsis thaliana.

Heat stress (HS) severely affects different cellular compartments operating in metabolic processes and represents a critical threat to plant growth and yield. Chloroplasts are crucial for heat stress response (HSR), signaling to the nucleus the environmental challenge and adjusting metabolic and biosynthetic functions accordingly. GENOMES UNCOUPLED 1 (GUN1), a chloroplast-localized protein, has been recognized as one of the main players of chloroplast retrograde signaling. Here, we investigate HSR in Arabidopsis wild-type and gun1 plantlets subjected to 2 hours of HS at 45°C. In wild-type plants, Reactive Oxygen Species (ROS) accumulate promptly after HS, contributing to transiently oxidize the cellular environment and acting as signaling molecules. After 3 hours of physiological recovery at growth temperature (22°C), the induction of enzymatic and non-enzymatic antioxidants prevents oxidative damage. On the other hand, gun1 mutants fail to induce the oxidative burst immediately after HS and accumulate ROS and oxidative damage after 3 hours of recovery at 22°C, thus resulting in enhanced sensitivity to HS. These data suggest that GUN1 is required to oxidize the cellular environment, participating in the acquisition of basal thermotolerance through the redox-dependent plastid-to-nucleus communication.

The PUB4 E3 Ubiquitin Ligase Is Responsible for the Variegated Phenotype Observed upon Alteration of Chloroplast Protein Homeostasis in Arabidopsis Cotyledons.

During a plant's life cycle, plastids undergo several modifications, from undifferentiated pro-plastids to either photosynthetically-active chloroplasts, ezioplasts, chromoplasts or storage organelles, such as amyloplasts, elaioplasts and proteinoplasts. Plastid proteome rearrangements and protein homeostasis, together with intracellular communication pathways, are key factors for correct plastid differentiation and functioning. When plastid development is affected, aberrant organelles are degraded and recycled in a process that involves plastid protein ubiquitination. In this study, we have analysed the Arabidopsis gun1-102 ftsh5-3 double mutant, lacking both the plastid-located protein GUN1 (Genomes Uncoupled 1), involved in plastid-to-nucleus communication, and the chloroplast-located FTSH5 (Filamentous temperature-sensitive H5), a metalloprotease with a role in photosystem repair and chloroplast biogenesis. gun1-102 ftsh5-3 seedlings show variegated cotyledons and true leaves that we attempted to suppress by introgressing second-site mutations in genes involved in: (i) plastid translation, (ii) plastid folding/import and (iii) cytosolic protein ubiquitination. Different phenotypic effects, ranging from seedling-lethality to partial or complete suppression of the variegated phenotype, were observed in the corresponding triple mutants. Our findings indicate that Plant U-Box 4 (PUB4) E3 ubiquitin ligase plays a major role in the target degradation of damaged chloroplasts and is the main contributor to the variegated phenotype observed in gun1-102 ftsh5-3 seedlings.

Gene dosage compensation of rRNA transcript levels in Arabidopsis thaliana lines with reduced ribosomal gene copy number.

The 45S rRNA genes (rDNA) are among the largest repetitive elements in eukaryotic genomes. rDNA consists of tandem arrays of rRNA genes, many of which are transcriptionally silenced. Silent rDNA repeats may act as 'back-up' copies for ribosome biogenesis and have nuclear organization roles. Through Cas9-mediated genome editing in the Arabidopsis thaliana female gametophyte, we reduced 45S rDNA copy number (CN) to a plateau of ∼10%. Two independent lines had rDNA CNs reduced by up to 90% at the T7 generation, named low copy number (LCN) lines. Despite drastic reduction of rDNA copies, rRNA transcriptional rates, and steady-state levels remained the same as wild-type plants. Gene dosage compensation of rRNA transcript levels was associated with reduction of silencing histone marks at rDNA loci and altered Nucleolar Organiser Region 2 organization. Although overall genome integrity of LCN lines appears unaffected, a chromosome segmental duplication occurred in one of the lines. Transcriptome analysis of LCN seedlings identified several shared dysregulated genes and pathways in both independent lines. Cas9 genome editing of rRNA repeats to generate LCN lines provides a powerful technique to elucidate rDNA dosage compensation mechanisms and impacts of low rDNA CN on genome stability, development, and cellular processes.

GUN1 and Plastid RNA Metabolism: Learning from Genetics.

GUN1 (genomes uncoupled 1), a chloroplast-localized pentatricopeptide repeat (PPR) protein with a C-terminal small mutS-related (SMR) domain, plays a central role in the retrograde communication of chloroplasts with the nucleus. This flow of information is required for the coordinated expression of plastid and nuclear genes, and it is essential for the correct development and functioning of chloroplasts. Multiple genetic and biochemical findings indicate that GUN1 is important for protein homeostasis in the chloroplast; however, a clear and unified view of GUN1's role in the chloroplast is still missing. Recently, GUN1 has been reported to modulate the activity of the nucleus-encoded plastid RNA polymerase (NEP) and modulate editing of plastid RNAs upon activation of retrograde communication, revealing a major role of GUN1 in plastid RNA metabolism. In this opinion article, we discuss the recently identified links between plastid RNA metabolism and retrograde signaling by providing a new and extended concept of GUN1 activity, which integrates the multitude of functional genetic interactions reported over the last decade with its primary role in plastid transcription and transcript editing.

NoPv1: a synthetic antimicrobial peptide aptamer targeting the causal agents of grapevine downy mildew and potato late blight.

Grapevine (Vitis vinifera L.) is a crop of major economic importance. However, grapevine yield is guaranteed by the massive use of pesticides to counteract pathogen infections. Under temperate-humid climate conditions, downy mildew is a primary threat for viticulture. Downy mildew is caused by the biotrophic oomycete Plasmopara viticola Berl. & de Toni, which can attack grapevine green tissues. In lack of treatments and with favourable weather conditions, downy mildew can devastate up to 75% of grape cultivation in one season and weaken newly born shoots, causing serious economic losses. Nevertheless, the repeated and massive use of some fungicides can lead to environmental pollution, negative impact on non-targeted organisms, development of resistance, residual toxicity and can foster human health concerns. In this manuscript, we provide an innovative approach to obtain specific pathogen protection for plants. By using the yeast two-hybrid approach and the P. viticola cellulose synthase 2 (PvCesA2), as target enzyme, we screened a combinatorial 8 amino acid peptide library with the aim to identify interacting peptides, potentially able to inhibit PvCesa2. Here, we demonstrate that the NoPv1 peptide aptamer prevents P. viticola germ tube formation and grapevine leaf infection without affecting the growth of non-target organisms and without being toxic for human cells. Furthermore, NoPv1 is also able to counteract Phytophthora infestans growth, the causal agent of late blight in potato and tomato, possibly as a consequence of the high amino acid sequence similarity between P. viticola and P. infestans cellulose synthase enzymes.

Barley's Second Spring as A Model Organism for Chloroplast Research.

Barley (Hordeum vulgare) has been widely used as a model crop for studying molecular and physiological processes such as chloroplast development and photosynthesis. During the second half of the 20th century, mutants such as albostrians led to the discovery of the nuclear-encoded, plastid-localized RNA polymerase and the retrograde (chloroplast-to-nucleus) signalling communication pathway, while chlorina-f2 and xantha mutants helped to shed light on the chlorophyll biosynthetic pathway, on the light-harvesting proteins and on the organization of the photosynthetic apparatus. However, during the last 30 years, a large fraction of chloroplast research has switched to the more "user-friendly" model species Arabidopsis thaliana, the first plant species whose genome was sequenced and published at the end of 2000. Despite its many advantages, Arabidopsis has some important limitations compared to barley, including the lack of a real canopy and the absence of the proplastid-to-chloroplast developmental gradient across the leaf blade. These features, together with the availability of large collections of natural genetic diversity and mutant populations for barley, a complete genome assembly and protocols for genetic transformation and gene editing, have relaunched barley as an ideal model species for chloroplast research. In this review, we provide an update on the genomics tools now available for barley, and review the biotechnological strategies reported to increase photosynthesis efficiency in model species, which deserve to be validated in barley.

HEBE, a novel positive regulator of senescence in Solanum lycopersicum.

Leaf senescence and plant aging are traits of great interest for breeders. Senescing cells undergo important physiological and biochemical changes, while cellular structures such as chloroplasts are degraded with dramatic metabolic consequences for the whole plant. The possibility of prolonging the photosynthetic ability of leaves could positively impact the plant's life span with benefits for biomass production and metabolite accumulation; plants with these characteristics display a stay-green phenotype. A group of plant transcription factors known as NAC play a pivotal role in controlling senescence: here we describe the involvement of the tomato NAC transcription factor Solyc12g036480, which transcript is present in leaves and floral buds. Since its silencing delays leaf senescence and prevents plants from ageing, we renamed Solyc12g0364 HEBE, for the Greek goddess of youth. In this manuscript we describe how HEB downregulation negatively affects the progression of senescence, resulting in changes in transcription of senescence-promoting genes, as well as the activity of enzymes involved in chlorophyll degradation, thereby explaining the stay-green phenotype.

The plastid transcription machinery and its coordination with the expression of nuclear genome: Plastid-Encoded Polymerase, Nuclear-Encoded Polymerase and the Genomes Uncoupled 1-mediated retrograde communication.

Plastid genes in higher plants are transcribed by at least two different RNA polymerases, the plastid-encoded RNA polymerase (PEP), a bacteria-like core enzyme whose subunits are encoded by plastid genes (rpoA, rpoB, rpoC1 and rpoC2), and the nuclear-encoded plastid RNA polymerase (NEP), a monomeric bacteriophage-type RNA polymerase. Both PEP and NEP enzymes are active in non-green plastids and in chloroplasts at all developmental stages. Their transcriptional activity is affected by endogenous and exogenous factors and requires a strict coordination within the plastid and with the nuclear gene expression machinery. This review focuses on the different molecular mechanisms underlying chloroplast transcription regulation and its coordination with the photosynthesis-associated nuclear genes (PhANGs) expression. Particular attention is given to the link between NEP and PEP activity and the GUN1- (Genomes Uncoupled 1) mediated chloroplast-to-nucleus retrograde communication with respect to the Δrpo adaptive response, i.e. the increased accumulation of NEP-dependent transcripts upon depletion of PEP activity, and the editing-level changes observed in NEP-dependent transcripts, including rpoB and rpoC1, in gun1 cotyledons after norflurazon or lincomycin treatment. The role of cytosolic preproteins and HSP90 chaperone as components of the GUN1-retrograde signalling pathway, when chloroplast biogenesis is inhibited in Arabidopsis cotyledons, is also discussed. This article is part of the theme issue 'Retrograde signalling from endosymbiotic organelles'.

Higher order photoprotection mutants reveal the importance of ΔpH-dependent photosynthesis-control in preventing light induced damage to both photosystem II and photosystem I.

Although light is essential for photosynthesis, when in excess, it may damage the photosynthetic apparatus, leading to a phenomenon known as photoinhibition. Photoinhibition was thought as a light-induced damage to photosystem II; however, it is now clear that even photosystem I may become very vulnerable to light. One main characteristic of light induced damage to photosystem II (PSII) is the increased turnover of the reaction center protein, D1: when rate of degradation exceeds the rate of synthesis, loss of PSII activity is observed. With respect to photosystem I (PSI), an excess of electrons, instead of an excess of light, may be very dangerous. Plants possess a number of mechanisms able to prevent, or limit, such damages by safe thermal dissipation of light energy (non-photochemical quenching, NPQ), slowing-down of electron transfer through the intersystem transport chain (photosynthesis-control, PSC) in co-operation with the Proton Gradient Regulation (PGR) proteins, PGR5 and PGRL1, collectively called as short-term photoprotection mechanisms, and the redistribution of light between photosystems, called state transitions (responsible of fluorescence quenching at PSII, qT), is superimposed to these short term photoprotective mechanisms. In this manuscript we have generated a number of higher order mutants by crossing genotypes carrying defects in each of the short-term photoprotection mechanisms, with the final aim to obtain a direct comparison of their role and efficiency in photoprotection. We found that mutants carrying a defect in the ΔpH-dependent photosynthesis-control are characterized by photoinhibition of both photosystems, irrespectively of whether PSBS-dependent NPQ or state transitions defects were present or not in the same individual, demonstrating the primary role of PSC in photoprotection. Moreover, mutants with a limited capability to develop a strong PSBS-dependent NPQ, were characterized by a high turnover of the D1 protein and high values of Y(NO), which might reflect energy quenching processes occurring within the PSII reaction center.

BPC transcription factors and a Polycomb Group protein confine the expression of the ovule identity gene SEEDSTICK in Arabidopsis.

The BASIC PENTACYSTEINE (BPC) GAGA (C-box) binding proteins belong to a small plant transcription factor family. We previously reported that class I BPCs bind directly to C-boxes in the SEEDSTICK (STK) promoter and the mutagenesis of these cis-elements affects STK expression in the flower. The MADS-domain factor SHORT VEGETATIVE PHASE (SVP) is another key regulator of STK. Direct binding of SVP to CArG-boxes in the STK promoter are required to repress its expression during the first stages of flower development. Here we show that class II BPCs directly interact with SVP and that MADS-domain binding sites in the STK promoter region are important for the correct spatial and temporal expression of this homeotic gene. Furthermore, we show that class I and class II BPCs act redundantly to repress STK expression in the flower, most likely by recruiting TERMINAL FLOWER 2/LIKE HETEROCHROMATIN PROTEIN 1 (TFL2/LHP1) and mediating the establishment and the maintenance of H3K27me3 repressive marks on DNA. We investigate the role of LHP1 in the regulation of STK expression. In addition to providing a better understanding of the role of BPC transcription factors in the regulation of STK expression, our results suggest the existence of a more general regulatory complex composed of BPCs, MADS-domain factors and Polycomb Repressive Complexes that co-operate to regulate gene expression in reproductive tissues. We believe that our data along with the molecular model described here could provide significant insights for a more comprehensive understanding of gene regulation in plants.

GUN1 influences the accumulation of NEP-dependent transcripts and chloroplast protein import in Arabidopsis cotyledons upon perturbation of chloroplast protein homeostasis.

Correct chloroplast development and function require co-ordinated expression of chloroplast and nuclear genes. This is achieved through chloroplast signals that modulate nuclear gene expression in accordance with the chloroplast's needs. Genetic evidence indicates that GUN1, a chloroplast-localized pentatricopeptide repeat (PPR) protein with a C-terminal Small MutS-Related (SMR) domain, is involved in integrating multiple developmental and stress-related signals in both young seedlings and adult leaves. Recently, GUN1 was found to interact physically with factors involved in chloroplast protein homeostasis, and with enzymes of tetrapyrrole biosynthesis in adult leaves that function in various retrograde signalling pathways. Here we show that following perturbation of chloroplast protein homeostasis: (i) by growth in lincomycin-containing medium; or (ii) in mutants defective in either the FtsH protease complex (ftsh), plastid ribosome activity (prps21-1 and prpl11-1) or plastid protein import and folding (cphsc70-1), GUN1 influences NEP-dependent transcript accumulation during cotyledon greening and also intervenes in chloroplast protein import.

Time-Course Transcriptome Analysis of Arabidopsis Siliques Discloses Genes Essential for Fruit Development and Maturation.

Fruits protect the developing seeds of angiosperms and actively contribute to seed dispersion. Furthermore, fruit and seed development are highly synchronized and require exchange of information between the mother plant and the developing generations. To explore the mechanisms controlling fruit formation and maturation, we performed a transcriptomic analysis on the valve tissue of the Arabidopsis (Arabidopsis thaliana) silique using RNA sequencing. In doing so, we have generated a data set of differentially regulated genes that will help to elucidate the molecular mechanisms that underpin the initial phase of fruit growth and, subsequently, trigger fruit maturation. The robustness of our data set has been tested by functional genomic studies. Using a reverse genetics approach, we selected 10 differentially expressed genes and explored the consequences of their disruption for both silique growth and senescence. We found that genes contained in our data set play essential roles in different stages of silique development and maturation, indicating that our transcriptome-based gene list is a powerful tool for the elucidation of the molecular mechanisms controlling fruit formation in Arabidopsis.

Trans-splicing of plastid rps12 transcripts, mediated by AtPPR4, is essential for embryo patterning in Arabidopsis thaliana.

MAIN CONCLUSION: AtPPR4-mediated trans-splicing of plastid rps12 transcripts is essential for key embryo morphogenetic events such as development of cotyledons, determination of provascular tissue, and organization of the shoot apical meristem (SAM), but not for the formation of the protodermal layer. Members of the pentatricopeptide repeat (PPR) containing protein family have emerged as key regulators of the organelle post-transcriptional processing and to be essential for proper plant embryo development. In this study, we report the functional characterization of the AtPPR4 (At5g04810) gene encoding a plastid nucleoid PPR protein. In-situ hybridization analysis reveals the presence of AtPPR4 transcripts already at the transition stage of embryo development. As a consequence, embryos lacking the AtPPR4 protein arrest their development at the transition/early-heart stages and show defects in the determination of the provascular tissue and organization of SAM. This complex phenotype is due to the specific role of AtPPR4 in the trans-splicing of the plastid rps12 transcripts, as shown by northern and slot-blot hybridizations, and the consequent defect in 70S ribosome accumulation and plastid protein synthesis, in agreement with the role proposed for the maize orthologue, ZmPPR4.

The DEAD-box RNA Helicase RH50 Is a 23S-4.5S rRNA Maturation Factor that Functionally Overlaps with the Plastid Signaling Factor GUN1.

DEAD-box RNA helicases (DBRHs) modulate RNA secondary structure, allowing RNA molecules to adopt the conformations required for interaction with their target proteins. RH50 is a chloroplast-located DBRH that colocalizes and is coexpressed with GUN1, a central factor in chloroplast-to-nucleus signaling. When combined with mutations that impair plastid gene expression (prors1-1, prpl11-1, prps1-1, prps21-1, prps17-1, and prpl24-1), rh50 and gun1 mutations evoke similar patterns of epistatic effects. These observations, together with the synergistic growth phenotype of the double mutant rh50-1 gun1-102, suggest that RH50 and GUN1 are functionally related and that this function is associated with plastid gene expression, in particular ribosome functioning. However, rh50-1 itself is not a gun mutant, although-like gun1-102-the rh50-1 mutation suppresses the down-regulation of nuclear genes for photosynthesis induced by the prors1-1 mutation. The RH50 protein comigrates with ribosomal particles, and is required for efficient translation of plastid proteins. RH50 binds to transcripts of the 23S-4.5S intergenic region and, in its absence, levels of the corresponding rRNA processing intermediate are strongly increased, implying that RH50 is required for the maturation of the 23S and 4.5S rRNAs. This inference is supported by the finding that loss of RH50 renders chloroplast protein synthesis sensitive to erythromycin and exposure to cold. Based on these results, we conclude that RH50 is a plastid rRNA maturation factor.

CRP1 Protein: (dis)similarities between Arabidopsis thaliana and Zea mays.

Biogenesis of chloroplasts in higher plants is initiated from proplastids, and involves a series of processes by which a plastid able to perform photosynthesis, to synthesize amino acids, lipids, and phytohormones is formed. All plastid protein complexes are composed of subunits encoded by the nucleus and chloroplast genomes, which require a coordinated gene expression to produce the correct concentrations of organellar proteins and to maintain organelle function. To achieve this, hundreds of nucleus-encoded factors are imported into the chloroplast to control plastid gene expression. Among these factors, members of the Pentatricopeptide Repeat (PPR) containing protein family have emerged as key regulators of the organellar post-transcriptional processing. PPR proteins represent a large family in plants, and the extent to which PPR functions are conserved between dicots and monocots deserves evaluation, in light of differences in photosynthetic metabolism (C3 vs. C4) and localization of chloroplast biogenesis (mesophyll vs. bundle sheath cells). In this work we investigated the role played in the process of chloroplast biogenesis by At5g42310, a member of the Arabidopsis PPR family which we here refer to as AtCRP1 (Chloroplast RNA Processing 1), providing a comparison with the orthologous ZmCRP1 protein from Zea mays. Loss-of-function atcrp1 mutants are characterized by yellow-albinotic cotyledons and leaves owing to defects in the accumulation of subunits of the thylakoid protein complexes. As in the case of ZmCRP1, AtCRP1 associates with the 5' UTRs of both psaC and, albeit very weakly, petA transcripts, indicating that the role of CRP1 as regulator of chloroplast protein synthesis has been conserved between maize and Arabidopsis. AtCRP1 also interacts with the petB-petD intergenic region and is required for the generation of petB and petD monocistronic RNAs. A similar role has been also attributed to ZmCRP1, although the direct interaction of ZmCRP1 with the petB-petD intergenic region has never been reported, which could indicate that AtCRP1 and ZmCRP1 differ, in part, in their plastid RNA targets.

Improved Drought Stress Response in Alfalfa Plants Nodulated by an IAA Over-producing Rhizobium Strain.

The drought-stress response in plant involves the cross-talk between abscisic acid (ABA) and other phytohormones, such as jasmonates and ethylene. The auxin indole-3-acetic acid (IAA) plays an integral part in plant adaptation to drought stress. Investigation was made to see how the main auxin IAA interacted with other plant hormones under water stress, applied through two different growth conditions (solid and hydroponic). Medicago sativa plants nodulated by the Ensifer meliloti wild type 1021 (Ms-1021) and its IAA-overproducing RD64 derivative strains (Ms-RD64) were subjected to drought stress, comparing their response. When the expression of nifH gene and the activity of the nitrogenase enzyme were measured after stress treatments, Ms-RD64 plants recorded a significantly weaker damage. These results were correlated with a lower biomass reduction, and a higher Rubisco protein level measured for the Ms-RD64-stressed plants as compared to the Ms-1021-stressed ones. It has been verified that the stress response observed for Ms-RD64-stressed plants was related to the production of greater amount of low-molecular-weight osmolytes, such as proline and pinitol, measured in these plants. For the Ms-RD64 plants the immunoblotting analysis of thylakoid membrane proteins showed that some of the photosystem proteins increased after the stress. An increased non-photochemical quenching after the stress was also observed for these plants. The reduced wilting signs observed for these plants were also connected to the significant down-regulation of the MtAA03 gene involved in the ABA biosynthesis, and with the unchanged expression of the two genes (Mt-2g006330 and Mt-8g095330) of ABA signaling. When the expression level of the ethylene-signaling genes was evaluated by qPCR analysis no significant alteration of the key positive regulators was recorded for Ms-RD64-stressed plants. Coherently, these plants accumulated 40% less ethylene as compared to Ms-1021-stressed ones. The results presented herein indicate that the variations in endogenous IAA levels, triggered by the overproduction of rhizobial IAA inside root nodules, positively affected drought stress response in nodulated alfalfa plants.