Plants respond to hypoxia, often caused by submergence, by expressing a specific set of genes that contribute to acclimation to this unfavorable environmental condition. Genes induced by low oxygen include those encoding enzymes for carbohydrate metabolism and fermentation, pathways that are required for survival. Sugar availability is therefore of crucial importance for energy production under hypoxia. Here, we show that Arabidopsis (Arabidopsis thaliana) plants require starch for surviving submergence as well as for ensuring the rapid induction of genes encoding enzymes required for anaerobic metabolism. The starchless pgm mutant is highly susceptible to submergence and also fails to induce anaerobic genes at the level of the wild type. Treating wild-type plants under conditions inducing sugar starvation results in a weak induction of alcohol dehydrogenase and other anaerobic genes. Induction of gene expression under hypoxia requires transcription factors belonging to group VII ethylene response factors (ERF-VII) that, together with plant Cys oxidases, act as an oxygen-sensing mechanism. We show that repression of this pathway by sugar starvation occurs downstream of the hypoxia-dependent stabilization of ERF-VII proteins and independently of the energy sensor protein kinases SnRK1.1 (SNF1-related kinase 1.1).

Summary: Low oxygen availability often is associated with soil waterlogging or submergence, but may occur also as hypoxic niches in otherwise aerobic tissues. Experimental evidence assigns a role in Botrytis cinerea resistance to a group of oxygen‐unstable Ethylene Response Factors (ERF‐VII). Given that infection by B. cinerea often occurs in aerobic organs such as leaves, where ERF‐VII stability should be compromised, we explored the possibility of local leaf hypoxia at the site of infection. We analyzed the expression of hypoxia‐responsive genes in infected leaves. Confocal microscopy was utilized to verify the localization of the ERF‐VII protein RAP2.12. Oxygen concentration was measured to evaluate the availability of oxygen (O₂). We discovered that infection by B. cinerea induces increased respiration, leading to a drastic drop in the O₂ concentration in an otherwise fully aerobic leaf. The establishment of a local hypoxic area results in stabilization and nuclear relocalization of RAP2.12. The possible roles of defence elicitors, ABA and ethylene were evaluated. Local hypoxia at the site of B. cinerea infection allows the stabilization of ERF‐VII proteins. Hypoxia at the site of pathogen infection generates a nearly O₂‐free environment that may affect the stability of other N‐degron‐regulated proteins as well as the metabolism of elicitors.

A comprehensive effort was devoted to exploring, collecting and characterizing the local Maltese olive germplasm, often represented by ancient, monumental trees and by plants of uncertain origin. SSR and cp-SSR analysis of all samples enabled the identification of 46 genotypes and establishment of the correspondence between ancient trees, main local varieties and other Mediterranean cultivars. The application of plastid markers enabled identification of two lineages among Maltese genotypes, with more than 50% represented by lineage E2. Twenty-nine cases of grafting were identified among the various genotypes and lineages. In most cases, E1 canopies were grafted on E2 rootstocks, but reverse cases were also observed. The phylogenetic study of Maltese genotypes, together with hundreds of cultivars from the Mediterranean Basin and beyond, highlights the richness of Maltese olive diversity and drawing attention to the genetic similarity of some Maltese olive genotypes with neighboring Italian and Algerian varieties. These results underline the long-lasting presence of the olive in the country, contributing to the reconstruction of its phylogeny and demonstrating a possible autochthonous origin of many samples. Some still-living ancient trees are at serious risk of extinction due to abandonment, urban expansion and environmental threats. This study supports the preservation of the Maltese olive germplasm and highlights its importance as a rich genetic source to face new agronomical challenges and future climatic constraints.

In plants, hypoxia can be induced by submergence, and the lack of oxygen impairs mitochondrial respiration, thus affecting the plant’s energy status. Hypoxia has major effects on gene expression; these changes induce key responses that help meet the needs of the stressed plant. However, little is known about the possible role of RNA signaling in the regulation of gene expression under limited oxygen availability. Here, we report the contribution of ARGONAUTE1 (AGO1) to hypoxia-induced gene regulation in Arabidopsis (Arabidopsis thaliana). Submergence induced changes in levels of the microRNAs miR2936 and miR398, but this had no obvious effects on their putative target mRNA levels. However, we found that ago1-27 plants are intolerant to submergence and transcriptome analysis identified genes whose regulation requires functional AGO1. Analysis of mutants affected in various branches of RNA signaling highlighted the convergence of AGO1 signaling with the AGO4-dependent RNA-directed DNA methylation (RdDM) pathway. AGO4-dependent RdDM represses the expression of HOMOLOG OF RPW8 4 (HR4) and alters its response to submergence. Remarkably, methylation of the second exon of HR4 is not only reduced in ago4-1 but also in plants overexpressing a constitutively stable version of the oxygen sensor RELATED TO APETALA2 12 (RAP2.12), indicating convergence of oxygen signaling with epigenetic regulation of gene expression. Therefore, our results identify a role for AGO1 and AGO4 RNA-silencing pathways in low-oxygen signaling in Arabidopsis.

Optimal plant growth performance requires that the presence and action of growth signals, such as gibberellins (GAs), are coordinated with the availability of photo-assimilates. Here, we studied the links between GA biosynthesis and carbon availability, and the subsequent effects on growth. We established that carbon availability, light and dark cues, and the circadian clock ensure the timing and magnitude of GA biosynthesis and that disruption of these factors results in reduced GA levels and expression of downstream genes. Carbon-dependent nighttime induction of gibberellin 3-beta-dioxygenase 1 (GA3ox1) was severely hampered when preceded by reduced daytime light availability, leading specifically to reduced bioactive GA₄ levels, and coinciding with a decline in leaf expansion rate during the night. We attributed this decline in leaf expansion mostly to reduced photo-assimilates. However, plants in which GA limitation was alleviated had significantly improved leaf expansion, demonstrating the relevance of GAs in growth control under varying carbon availability. Carbon-dependent expression of upstream GA biosynthesis genes (Kaurene synthase and gibberellin 20 oxidase 1, GA20ox1) was not translated into metabolite changes within this short timeframe. We propose a model in which the extent of nighttime biosynthesis of bioactive GA₄ by GA3ox1 is determined by nighttime consumption of starch reserves, thus providing day-to-day adjustments of GA responses.

Exposure to high light intensity (HL) and cold treatment (CT) induces reddish pigmentation in Azolla filiculoides, an aquatic fern. Nevertheless, how these conditions, alone or in combination, influence Azolla growth and pigment synthesis remains to be fully elucidated. Likewise, the regulatory network underpinning the accumulation of flavonoids in ferns is still unclear. Here, we grew A. filiculoides under HL and/or CT conditions for 20 days and evaluated the biomass doubling time, relative growth rate, photosynthetic and non-photosynthetic pigment contents, and photosynthetic efficiency by chlorophyll fluorescence measurements. Furthermore, from the A. filiculoides genome, we mined the homologs of MYB, bHLH, and WDR genes, which form the MBW flavonoid regulatory complex in higher plants, to investigate their expression by qRT-PCR. We report that A. filiculoides optimizes photosynthesis at lower light intensities, regardless of the temperature. In addition, we show that CT does not severely hamper Azolla growth, although it causes the onset of photoinhibition. Coupling CT with HL stimulates the accumulation of flavonoids, which likely prevents irreversible photoinhibition-induced damage. Although our data do not support the formation of MBW complexes, we identified candidate MYB and bHLH regulators of flavonoids. Overall, the present findings are of fundamental and pragmatic relevance to Azolla’s biology.