As non-photosynthesizing organs, roots are dependent on diffusion of oxygen from the external environment and, in some instances, from the shoot for their aerobic metabolism. Establishment of hypoxic niches in the developing tissues of plants has been postulated as a consequence of insufficient diffusion of oxygen to satisfy the demands throughout development. Here, we report that such niches are established at specific stages of lateral root primordia development in Arabidopsis thaliana grown under aerobic conditions. Using gain-and loss-of-function mutants, we show that ERF-VII transcription factors, which mediate hypoxic responses, control root architecture by acting in cells with a high level of auxin signaling. ERF-VIIs repress the expression of the auxin-induced genes LBD16, LBD18, and PUCHI, which are essential for lateral root development, by binding to their promoters. Our results support a model in which the establishment of hypoxic niches in the developing lateral root primordia contributes to the shutting down of key auxin-induced genes and regulates the production of lateral roots.

In higher plants, molecular responses to exogenous hypoxia are driven by group VII ethylene response factors (ERF-VIIs). These transcriptional regulators accumulate in the nucleus under hypoxia to activate anaerobic genes but are destabilized in normoxic conditions through the action of oxygen-sensing plant cysteine oxidases (PCOs). The PCOs catalyze the reaction of oxygen with the conserved N-terminal cysteine of ERF-VIIs to form cysteine sulfinic acid, triggering degradation via the Cys/Arg branch of the N-degron pathway. The PCOs are therefore a vital component of the plant oxygen signaling system, connecting environmental stimulus with cellular and physiological response. Rational manipulation of PCO activity could regulate ERF-VII levels and improve flood tolerance, but requires detailed structural information. We report crystal structures of the constitutively expressed PCO4 and PCO5 from Arabidopsis thaliana to 1.24 and 1.91 Å resolution, respectively. The structures reveal that the PCOs comprise a cupin-like scaffold, which supports a central metal cofactor coordinated by three histidines. While this overall structure is consistent with other thiol dioxygenases, closer inspection of the active site indicates that other catalytic features are not conserved, suggesting that the PCOs may use divergent mechanisms to oxidize their substrates. Conservative substitution of two active site residues had dramatic effects on PCO4 function both in vitro and in vivo, through yeast and plant complementation assays. Collectively, our data identify key structural elements that are required for PCO activity and provide a platform for engineering crops with improved hypoxia tolerance.

Alcohol dehydrogenase (ADH) and pyruvate decarboxylase (PDC) are key to the establishment of the fermentative metabolism in plants during oxygen shortage. Most of the evidence that both ADH and PDC are required for plant tolerance to hypoxia comes from experiments performed by limiting oxygen in the environment, such as by exposing plants to gaseous hypoxia or to waterlogging or submergence. However, recent experiments have shown that hypoxic niches might exist in plants grown in aerobic conditions. Here, we investigated the importance of ADH and PDC for plant growth and development under aerobic conditions, long-term waterlogging and short-term submergence. Data were collected after optimizing the software associated with a commercially-available phenotyping instrument, to circumvent problems in separation of plants and background pixels based on colour features, which is not applicable for low-oxygen stressed plants due to the low colour contrast of leaves with the brownish soil. The results showed that the growth penalty associated with the lack of functional ADH1 or both PDC1 and PDC2 is greater under aerobic conditions than in hypoxia, highlighting the importance of fermentative metabolism in plants grown under normal, aerobic conditions.

Plants seem to take up exogenous RNA that was artificially designed to target specific genes, followed by activation of the RNA interference (RNAi) machinery. It is, however, not known whether plants use RNAs themselves as signalling molecules in plant-to-plant communication, other than evidence that an exchange of small RNAs occurs between parasitic plants and their hosts. Exogenous RNAs from the environment, if taken up by some living organisms, can indeed induce RNAi. This phenomenon has been observed in nematodes and insects, and host Arabidopsis cells secrete exosome-like extracellular vesicles to deliver plant small RNAs into Botrytis cinerea. Here we show that micro-RNAs (miRNAs) produced by plants act as signalling molecules affecting gene expression in other, nearby plants. Exogenous miRNAs, such as miR156 and miR399, trigger RNAi via a mechanism requiring both AGO1 and RDR6. This emphasizes that the production of secondary small interfering RNAs is required. This evidence highlights the existence of a mechanism in which miRNAs represent signalling molecules that enable communication between plants.

Due to the involvement of oxygen in many essential metabolic reactions, all living organisms have developed molecular systems that allow adaptive physiological and metabolic transitions depending on oxygen availability. In mammals, the expression of hypoxia-response genes is controlled by the heterodimeric Hypoxia-Inducible Factor. The activity of this transcriptional regulator is linked mainly to the oxygen-dependent hydroxylation of conserved proline residues in its a-subunit, carried out by prolyl-hydroxylases, and subsequent ubiquitination via the E3 ligase von Hippel-Lindau tumor suppressor, which targets Hypoxia-Inducible Factor-a to the proteasome. By exploiting bioengineered versions of this mammalian oxygen sensor, we designed and optimized a synthetic device that drives gene expression in an oxygen-dependent fashion in plants. Transient assays in Arabidopsis (Arabidopsis thaliana) mesophyll protoplasts indicated that a combination of the yeast Gal4/upstream activating sequence system and the mammalian oxygen sensor machinery can be used effectively to engineer a modular, oxygen-inducible transcriptional regulator. This synthetic device also was shown to be selectively controlled by oxygen in whole plants when its components were expressed stably in Arabidopsis seedlings. We envision the exploitation of our genetically encoded controllers to generate plants able to switch gene expression selectively depending on oxygen availability, thereby providing a proof of concept for the potential of synthetic biology to assist agricultural practices in environments with variable oxygen provision.