Rapid advances in microscopy have boosted research on cell biology. However sample preparation enabling excellent reproducible tissue preservation and cell labeling for in depth microscopic analysis of inner cell layers, tissues and organs still represents a major challenge for immunolocalization studies. Here we describe a protocol for whole-mount immunolocalization of proteins which is applicable to a wide range of plant species. The protocol is improved and robust for optimal sample fixation, tissue clearing and multi-protein staining procedures and can be used in combination with simultaneous detection of specific sequences of nucleic acids. In addition, cell wall and nucleus labelling can be implemented in the protocol, thereby allowing a detailed analysis of morphology and gene expression patterns with single-cell resolution. Besides enabling accurate, high resolution and reproducible protein detection in expression and localization studies, the procedure takes a single working day to complete without the need for robotic equipment.

Summary: The plant hormone auxin is a mobile signal which affects nuclear transcription by regulating the stability of auxin/indole‐3‐acetic acid (IAA) repressor proteins. Auxin is transported polarly from cell to cell by auxin efflux proteins of the PIN family, but it is not as yet clear how auxin levels are regulated within cells and how access of auxin to the nucleus may be controlled. The Arabidopsis genome contains eight PINs, encoding proteins with a similar membrane topology. While five of the PINs are typically targeted polarly to the plasma membranes, the smallest members of the family, PIN5 and PIN8, seem to be located not at the plasma membrane but in endomembranes. Here we demonstrate by electron microscopy analysis that PIN8, which is specifically expressed in pollen, resides in the endoplasmic reticulum and that it remains internally localized during pollen tube growth. Transgenic Arabidopsis and tobacco plants were generated overexpressing or ectopically expressing functional PIN8, and its role in control of auxin homeostasis was studied. PIN8 ectopic expression resulted in strong auxin‐related phenotypes. The severity of phenotypes depended on PIN8 protein levels, suggesting a rate‐limiting activity for PIN8. The observed phenotypes correlated with elevated levels of free IAA and ester‐conjugated IAA. Activation of the auxin‐regulated synthetic DR5 promoter and of auxin response genes was strongly repressed in seedlings overexpressing PIN8 when exposed to 1‐naphthalene acetic acid. Thus, our data show a functional role for endoplasmic reticulum‐localized PIN8 and suggest a mechanism whereby PIN8 controls auxin thresholds and access of auxin to the nucleus, thereby regulating auxin‐dependent transcriptional activity.

Apple (Malus domestica) fruitlet abscission represents an interesting model system to study the early phases of the shedding process, during which major transcriptomic changes and metabolic rearrangements occur within the fruit. In apple, the drop of fruits at different positions within the cluster can be selectively magnified through chemical thinners, such as benzyladenine and metamitron, acting as abscission enhancers. In this study, different abscission potentials were obtained within the apple fruitlet population by means of the above-cited thinners. A metabolomic study was conducted on the volatile organic compounds emitted by abscising fruitlets, allowing for identification of isoprene as an early marker of abscission induction. A strong correlation was also observed between isoprene production and abscisic acid (ABA) levels in the fruit cortex, which were shown to increase in abscising fruitlets with respect to nonabscising ones. Transcriptomic evidence indicated that abscission-related ABA is biologically active, and its increased biosynthesis is associated with the induction of a specific ABA-responsive 9-cis-epoxycarotenoid dioxygenase gene. According to a hypothetical model, ABA may transiently cooperate with other hormones and secondary messengers in the generation of an intrafruit signal leading to the downstream activation of the abscission zone. The shedding process therefore appears to be triggered by multiple interdependent pathways, whose fine regulation, exerted within a very short temporal window by both endogenous and exogenous factors, determines the final destiny of the fruitlets.

Abscission has significant implications in agriculture and several efforts have been addressed by researchers to understand its regulatory steps in both model and crop species. Among the main players in abscission, ethylene has exhibited some fascinating features, in that it was shown to be involved at different stages of abscission induction and, in some cases, with interesting roles also within the abscising organ at the very early stages of the process. This review summarizes the current knowledge about the role of ethylene both at the level of the abscission zone and within the shedding organ, pointing out the missing pieces of the very complicated puzzle of the abscission process in the different species.

Apple (Malus×domestica Borkh) fruits are stored for long periods of time at low temperatures (1 °C) leading to the occurrence of physiological disorders. ‘Superficial scald’ of Granny Smith apples, an economically important ethylene-dependent disorder, was used as a model to study relationships among ethylene action, the regulation of the ROP-GAP rheostat, and maintenance of H₂O₂ homeostasis in fruits during prolonged cold exposure. The ROP-GAP rheostat is a key module for adaptation to low oxygen in Arabidopsis through Respiratory Burst NADPH Oxidase Homologs (RBOH)-mediated and ROP GTPase-dependent regulation of reactive oxygen species (ROS) homeostasis. Here, it was shown that the transcriptional expression of several components of the apple ROP-GAP machinery, including genes encoding RBOHs, ROPs, and their ancillary proteins ROP-GEFs and ROP-GAPs, is coordinately and negatively regulated by ethylene in conjunction with the progressive impairment of apoplastic H₂O₂ homeostatic levels. RNA sequencing analyses showed that several components of the known ROP- and ROS-associated transcriptional networks are regulated along with the ROP-GAP rheostat in response to ethylene perception. These findings may extend the role of the ROP-GAP rheostat beyond hypoxic responses and suggest that it may be a functional regulatory node involved in the integration of ethylene and ROS signalling pathways in abiotic stress.

Plants respond to gravitational force through directional growth along the gravity vector. Although auxin is the central component of the root graviresponse, it works in concert with other plant hormones. Here, we show that the folate precursor para-aminobenzoic acid (PABA) is a key modulator of the auxin-ethylene interplay during root gravitropism in Arabidopsis (Arabidopsis thaliana). In gravistimulated roots, PABA promotes an asymmetric auxin response, which causes the asymmetric growth responsible for root curvature. This activity requires the auxin response transcription factors AUXIN RESPONSE FACTOR7 (ARF7) and ARF19 as well as ethylene biosynthesis and signaling, indicating that PABA activity requires both auxin and ethylene pathways. Similar to ethylene, exogenous PABA reverses the agravitropic root growth of the auxin transport mutant pin-formed2 (pin2) and the auxin biosynthetic double mutant with loss of function of weak ethylene insensitive (wei) genes, wei8wei2, but not the pin2wei8wei2 triple mutant. This finding suggests that PABA regulates the ethylene-dependent reciprocal compensation between auxin transport and biosynthesis. Furthermore, manipulation of endogenous free PABA levels by modulating the expression of the gene encoding its glucosylation enzyme, UDP-GLYCOSYL TRANSFERASE75B1, impacts the root graviresponse, suggesting that endogenous free PABA levels may play a crucial role in modulating the auxin-ethylene cross talk necessary for root gravitropism.