Nitric oxide and cytokinin cross-talk and their role in plant hypoxia response.

Nitric oxide (NO) and cytokinins (CKs) are known for their crucial contributions to plant development, growth, senescence, and stress response. Despite the importance of both signals in stress responses, their interaction remains largely unexplored. The interplay between NO and CKs emerges as particularly significant not only regarding plant growth and development but also in addressing plant stress response, particularly in the context of extreme weather events leading to yield loss. In this review, we summarize NO and CKs metabolism and signaling. Additionally, we emphasize the crosstalk between NO and CKs, underscoring its potential impact on stress response, with a focus on hypoxia tolerance. Finally, we address the most urgent questions that demand answers and offer recommendations for future research endeavors.

Reciprocal modulation of responses to nitrate starvation and hypoxia in roots and leaves of Arabidopsis thaliana.

The flooding of agricultural land leads to hypoxia and nitrate leaching. While understanding the plant's response to these conditions is essential for crop improvement, the effect of extended nitrate limitation on subsequent hypoxia has not been studied in an organ-specific manner. We cultivated Arabidopsis thaliana without nitrate for 1 week before inducing hypoxia by bubbling the hydroponic solution with nitrogen gas for 16 h. In the roots, the transcripts of two transcription factor genes (HRA1, HRE2) and three genes involved in fermentation (SUS4, PDC1, ADH1) were ~10- to 100-fold upregulated by simultaneous hypoxia and nitrate starvation compared to the control condition (replete nitrate and oxygen). In contrast, this hypoxic upregulation was ~5 to 10 times stronger when nitrate was available. The phytoglobin genes PGB1 and PGB2, involved in nitric oxide (NO) scavenging, were massively downregulated by nitrate starvation (~1000-fold and 105-fold, respectively), but only under ambient oxygen levels; this was reflected in a 2.5-fold increase in NO concentration. In the leaves, HRA1, SUS4, and RAP2.3 were upregulated ~20-fold by hypoxia under nitrate starvation, whereas this upregulation was virtually absent in the presence of nitrate. Our results highlight that the plant's responses to nitrate starvation and hypoxia can influence each other.

Cross-stress tolerance: Mild nitrogen (N) deficiency effects on drought stress response of tomato (Solanum lycopersicum L.).

Climate change will lead to more frequent and severe drought periods which massively reduce crop production worldwide. Besides drought, nitrogen (N)-deficiency is another critical threat to crop yield production. Drought and N-deficiency both decrease photosynthesis and induce similar adaptive strategies such as longer roots, reduction of biomass, induction of reactive oxygen species (ROS), and antioxidative enzymes. Due to the overlapping response to N-deficiency and drought, understanding the physiological and molecular mechanisms involved in cross-stresses tolerance is crucial for breeding strategies and achieving multiple stress resistance and eventually more sustainable agriculture. The objective of this study was to investigate the effect of a mild N-deficiency on drought stress tolerance of tomato plants (Solanum lycopersicum L., cv. Moneymaker). Various morphological and physiological parameters such as dry biomass, root length, water potential, SPAD values, stomatal conductance, and compatible solutes accumulation (proline and sugar) were analyzed. Moreover, the expression of ROS scavenging marker genes, cytosolic ASCORBATE PEROXIDASES (cAPX1, cAPX2, and cAPX3), were investigated. Our results showed that a former mild N-deficiency (2 mM NO3 -) enhances plant adaptive response to drought stress (4 days) when compared to the plants treated with adequate N (5 mM NO3 -). The improved adaptive response was reflected in higher aboveground biomass, longer root, increased specific leaf weight, enhanced stomatal conductance (without reducing water content), and higher leaf sugar content. Moreover, the APX1 gene showed a higher expression level compared to control under N-deficiency and in combination with drought in the leaf, after a one-week recovery period. Our finding highlights a potentially positive link between a former mild N-deficiency and subsequent drought stress response in tomato. Combining the morphological and physiological response with underlying gene regulatory networks under consecutive stress, provide a powerful tool for improving multiple stress resistance in tomato which can be further transferred to other economically important crops.

Author Correction: RNA-Seq reveals novel genes and pathways associated with hypoxia duration and tolerance in tomato root.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Identification of nitric oxide (NO)-responsive genes under hypoxia in tomato (Solanum lycopersicum L.) root.

Flooding periods, as one probable consequence of climate change, will lead more frequently to plant hypoxic stress. Hypoxia sensing and signaling in the root, as the first organ encountering low oxygen, is therefore crucial for plant survival under flooding. Nitric oxide has been shown to be one of the main players involved in hypoxia signaling through the regulation of ERFVII transcription factors stability. Using SNP as NO donor, we investigated the NO-responsive genes, which showed a significant response to hypoxia. We identified 395 genes being differentially regulated under both hypoxia and SNP-treatment. Among them, 251 genes showed up- or down-regulation under both conditions which were used for further biological analysis. Functional classification of these genes showed that they belong to different biological categories such as primary carbon and nitrogen metabolism (e.g. glycolysis, fermentation, protein and amino acid metabolism), nutrient and metabolites transport, redox homeostasis, hormone metabolism, regulation of transcription as well as response to biotic and abiotic stresses. Our data shed light on the NO-mediated gene expression modulation under hypoxia and provides potential targets playing a role in hypoxia tolerance. These genes are interesting candidates for further investigating their role in hypoxia signaling and survival.

RNA-Seq reveals novel genes and pathways associated with hypoxia duration and tolerance in tomato root.

Due to climate change, economically important crop plants will encounter flooding periods causing hypoxic stress more frequently. This may lead to reduced yields and endanger food security. As roots are the first organ to be affected by hypoxia, the ability to sense and respond to hypoxic stress is crucial. At the molecular level, therefore, fine-tuning the regulation of gene expression in the root is essential for hypoxia tolerance. Using an RNA-Seq approach, we investigated transcriptome modulation in tomato roots of the cultivar 'Moneymaker', in response to short- (6 h) and long-term (48 h) hypoxia. Hypoxia duration appeared to have a significant impact on gene expression such that the roots of five weeks old tomato plants showed a distinct time-dependent transcriptome response. We observed expression changes in 267 and 1421 genes under short- and long-term hypoxia, respectively. Among these, 243 genes experienced changed expression at both time points. We identified tomato genes with a potential role in aerenchyma formation which facilitates oxygen transport and may act as an escape mechanism enabling hypoxia tolerance. Moreover, we identified differentially regulated genes related to carbon and amino acid metabolism and redox homeostasis. Of particular interest were the differentially regulated transcription factors, which act as master regulators of downstream target genes involved in responses to short and/or long-term hypoxia. Our data suggest a temporal metabolic and anatomic adjustment to hypoxia in tomato root which requires further investigation. We propose that the regulated genes identified in this study are good candidates for further studies regarding hypoxia tolerance in tomato or other crops.

Divergent N Deficiency-Dependent Senescence and Transcriptome Response in Developmentally Old and Young Brassica napus Leaves.

In the spring oilseed rape (OSR) cultivar 'Mozart' grown under optimal N supply (NO) or mild N deficiency (NL) the transcriptome changes associated with progressing age until early senescence in developmentally old lower canopy leaves (leaf #4) and younger higher canopy leaves (leaf #8) were investigated. Twelve weeks old NO and NL plants appeared phenotypically and transcriptomically identical, but thereafter distinct nutrition-dependent differences in gene expression patterns in lower and upper canopy leaves emerged. In NO leaves #4 of 14-week-old compared to 13-week-old plants, ∼600 genes were up- or downregulated, whereas in NL leaves #4 ∼3000 genes were up- or downregulated. In contrast, in 15-week-old compared to 13-week-old upper canopy leaves #8 more genes were up- or downregulated in optimally N-supplied plants (∼2000 genes) than in N-depleted plants (∼750 genes). This opposing effect of N depletion on gene regulation was even more prominent among photosynthesis-related genes (PSGs). Between week 13 and 14 in leaves #4, 99 of 110 PSGs were downregulated in NL plants, but none in NO plants. In contrast, from weeks 13 to 16 in leaves #8 of NL plants only 11 PSGs were downregulated in comparison to 66 PSGs in NO plants. Different effects of N depletion in lower versus upper canopy leaves were also apparent in upregulation of autophagy genes and NAC transcription factors. More than half of the regulated NAC and WRKY transcription factor, autophagy and protease genes were specifically regulated in NL leaves #4 or NO leaves #8 and thus may contribute to differences in senescence and nutrient mobilization in these leaves. We suggest that in N-deficient plants the upper leaves retain their N resources longer than in amply fertilized plants and remobilize them only after shedding of the lower leaves.

Strigolactone biosynthesis is evolutionarily conserved, regulated by phosphate starvation and contributes to resistance against phytopathogenic fungi in a moss, Physcomitrella patens.

In seed plants, strigolactones (SLs) regulate architecture and induce mycorrhizal symbiosis in response to environmental cues. SLs are formed by combined activity of the carotenoid cleavage dioxygenases (CCDs) 7 and 8 from 9-cis-ß-carotene, leading to carlactone that is converted by cytochromes P450 (clade 711; MAX1 in Arabidopsis) into various SLs. As Physcomitrella patens possesses CCD7 and CCD8 homologs but lacks MAX1, we investigated if PpCCD7 together with PpCCD8 form carlactone and how deletion of these enzymes influences growth and interactions with the environment. We investigated the enzymatic activity of PpCCD7 and PpCCD8 in vitro, identified the formed products by high performance liquid chromatography (HPLC) and LC-MS, and generated and analysed ΔCCD7 and ΔCCD8 mutants. We defined enzymatic activity of PpCCD7 as a stereospecific 9-cis-CCD and PpCCD8 as a carlactone synthase. ΔCCD7 and ΔCCD8 lines showed enhanced caulonema growth, which was revertible by adding the SL analogue GR24 or carlactone. Wild-type (WT) exudates induced seed germination in Orobanche ramosa. This activity was increased upon phosphate starvation and abolished in exudates of both mutants. Furthermore, both mutants showed increased susceptibility to phytopathogenic fungi. Our study reveals the deep evolutionary conservation of SL biosynthesis, SL function, and its regulation by biotic and abiotic cues.