Protocol: An improved and universal procedure for whole-mount immunolocalization in plants

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

A Grapevine TTG2-Like WRKY Transcription Factor Is Involved in Regulating Vacuolar Transport and Flavonoid Biosynthesis

open7noA small set of TTG2-like homolog proteins from different species belonging to the WRKY family of transcription factors were shown to share a similar mechanism of action and to control partially conserved biochemical/developmental processes in their native species. In particular, by activating P-ATPases residing on the tonoplast, PH3 from Petunia hybrida promotes vacuolar acidification in petal epidermal cells whereas TTG2 from Arabidopsis thaliana enables the accumulation of proanthocyanidins in the seed coat. In this work we functionally characterized VvWRKY26 identified as the closest grapevine homolog of PhPH3 and AtTTG2. When constitutively expressed in petunia ph3 mutant, VvWRKY26 can fulfill the PH3 function in the regulation of vacuolar pH and restores the wild type pigmentation phenotype. By a global correlation analysis of gene expression and by transient over-expression in Vitis vinifera, we showed transcriptomic relationships of VvWRKY26 with many genes related to vacuolar acidification and transport in grapevine. Moreover, our results indicate an involvement in flavonoid pathway possibly restricted to the control of proanthocyanidin biosynthesis that is consistent with its expression pattern in grape berry tissues. Overall, the results show that, in addition to regulative mechanisms and biological roles shared with TTG2-like orthologs, VvWRKY26 can play roles in fleshy fruit development that have not been previously reported in studies from dry fruit species. This study paves the way toward the comprehension of the regulatory network controlling vacuolar acidification and flavonoid accumulation mechanisms that contribute to the final berry quality traits in grapevine.openAmato, Alessandra; Cavallini, Erika; Zenoni, Sara; Finezzo, Laura; Begheldo, Maura; Ruperti, Benedetto; Tornielli, Giovanni BattistaAmato, Alessandra; Cavallini, Erika; Zenoni, Sara; Finezzo, Laura; Begheldo, Maura; Ruperti, Benedetto; Tornielli, Giovanni Battist

Ethylene negatively regulates transcript abundance of ROP-GAP rheostat-encoding genes and affects apoplastic reactive oxygen species homeostasis in epicarps of cold stored apple fruits

Apple (Malus 7domestica Borkh) fruits are stored for long periods of time at low temperatures (1 \ub0C) 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

Transcriptome profiling of ripening nectarine (Prunus persica L. Batsch) fruit treated with 1-MCP

A large-scale transcriptome analysis has been conducted using μPEACH1.0 microarray on nectarine (Prunus persica L. Batsch) fruit treated with 1-methylcyclopropene (1-MCP). 1-MCP maintained flesh firmness but did not block ethylene biosynthesis. Compared with samples at harvest, only nine genes appeared to be differentially expressed when fruit were sampled immediately after treatment, while a total of 90 targets were up- or down-regulated in untreated fruit. The effect of 1-MCP was confirmed by a direct comparison of transcript profiles in treated and untreated fruit after 24 h of incubation with 106 targets differentially expressed. About 30% of these targets correspond to genes involved in primary metabolism and response processes related to ethylene, auxin, and other hormones. In treated fruit, altered transcript accumulation was detected for some genes with a role in ripening-related events such as softening, colour development, and sugar metabolism. A rapid decrease in flesh firmness and an increase in ethylene production were observed in treated fruit maintained for 48 h in air at 20 °C after the end of the incubation period. Microarray comparison of this sample with untreated fruit 24 h after harvest revealed that about 45% of the genes affected by 1-MCP at the end of the incubation period changed their expression during the following 48 h in air. Among these genes, an ethylene receptor (ETR2) and three ethylene-responsive factors (ERF) were present, together with other transcription factors and ethylene-dependent genes involved in quality parameter changes

Ethylene and peach fruit ripening: a functional genomics approach

Fruit quality traits are the result of genetic, agronomic and environmental factors that, alone or in combination, modulate metabolic processes during both pre- and post-harvest phases and affect fruit development and ripening processes. Productivity, size and organoleptic quality should be the main quality criteria adopted by fruit growers: in this context, harvesting time is crucial. Too early harvested fruit may be stored for a long time but their flavour quality is low, whereas late harvested fruit are of better quality but do not withstand long storage periods and theirs shelf-life is reduced. This is particularly true for peaches and nectarines characterized by a quick ripening evolution and a reduced aptitude to prolonged storage: this induces growers to anticipate harvesting and represents the main constraints for supplying high-quality standard level peaches to the consumers. Elucidating mechanisms and basic processes characterizing ripening and responsible for the evolution of quality parameters is the prerequisite to develop strategies aimed to produce high-quality fruit and to maintain these standards throughout the postharvest chain. In climacteric fruit, including peaches, ethylene is a key factor in coordinating and regulating the evolution of several processes characterizing the ripening syndrome. Thus, studying aspects related to ethylene action has been a challenge during the last few decades. Improvements of the basic knowledge of ethylene physiology also came from the identification of specific inhibitors of its biosynthesis and/or action, and from the use of mutants. The development of highthroughput molecular tools (i.e. microarray) and functional genomics approaches represent a great opportunity for a better understanding of the ripening process and the basic mechanisms governing quality-related metabolisms in fruit. Considering peach, the first step toward a functional approach is represented by the development of an Expressed Sequences Tag (ESTs) repertoire, that, together with other ESTs isolated by other units and available in public databases, allowed to select 4806 oligos, corresponding to an equal number of genes expressed in peach fruit, and construct the first peach microarray (?PEACH 1.0). ?PEACH 1.0 has been used to study the effects of exogenous ethylene on different peach genotypes, a "melting flesh" cv and two ripening mutants, Slow Ripening (SR) and Stony Hard (SH). Microarray analysis revealed marked differences in transcript profiling possibly related to the nature of mutation and differences in ethylene physiology. SH fruit has also been used for expression analyses of two elements involved in the ethylene signalling pathway. Besides mutants, specific inhibitors of ethylene biosynthesis and/or action represent invaluable tools for elucidating the ethylene role in the ripening process. One of the most powerful inhibitor of ethylene function is 1-methylcyclopropene (1-MCP) that is practically used on different fruit species, but not on peaches, to prolong shelf-life. Using ?PEACH 1.0, a large-scale analysis of transcriptome has been performed on nectarine fruit treated with 1-MCP in order to elucidate the molecular mechanisms responsible for the limited effect of the inhibitor on this climacteric fruit species. At the end of the treatment (24h) and 48h hours later, a number of genes involved in quality-related ripening processes (such as softening, sugar metabolism and colour development) appeared to be deeply modified in their expression. Changes in the expression profile of Transcription Factors related to ethylene and auxin action confirmed the importance of "cross-talk" between the two hormones in the modulation of the ripening process in peach. In the context of a functional genomics, three different genes (two from peach and one from tomato), identified following transcriptomics approaches, have been used for transgenic experiments in tomato plants

Different postharvest conditions modulate ripening and ethylene biosynthetic and signal transduction pathways in Stony Hard peaches

Stony hard (SH) peaches are characterized, at ripening, by the maintenance of flesh firmness and the lack of ethylene production due to a reduced expression of Pp-ACS1. In a trial comparing melting flesh (MF, cv. ‘Summer Rich’) and SH (‘IFF331’ selection) fruit at two different postharvest temperatures (10 and 20 °C), unexpected behaviour was observed in SH peaches that displayed an increase in ethylene production and a decrease in flesh firmness when stored at 10 °C, a temperature regime basically ineffective in delaying ripening in MF fruit. This appeared to be the result of an induction of Pp-ACS1 transcription, making this genotype of particular interest for studying temperature stress physiology and ethylene-related ripening processes in peaches. Comparative expression analyses of genes involved in cell wall metabolism pointed out the presence of a negative (Pp-EG4), positive (Pp-endoPG) or no (one member of the PL family) relationship with ethylene at ripening. Results clearly showed that the last stage of firmness decrease (melting) only occurs in fruit producing ethylene and is associated with Pp-endoPG transcript accumulation. The expression of genes involved in ethylene biosynthesis and signalling pathways was evaluated using QRT-PCR. Pp-ACO1 appeared to be induced in SH kept at 10 °C but not at 20 °C. Transient increases in Pp-CTR1 and Pp-EIN2like gene expression have only been detected at the early stages of ripening in samples producing ethylene, indicating that a causal relationship might exist between ethylene and elements of its transduction pathway during peach fruit ripening

Different postharvest conditions modulate ripening and ethylene biosynthetic and signal transduction pathways in Stony Hard peaches

Stony hard (SH) peaches are characterized, at ripening, by the maintenance of flesh firmness and the lack of ethylene production due to a reduced expression of Pp-ACS1. In a trial comparing melting flesh (MF, cv. 'Summer Rich') and SH ('IFF331' selection) fruit at two different postharvest temperatures (10 and 20 °C), unexpected behaviour was observed in SH peaches that displayed an increase in ethylene production and a decrease in flesh firmness when stored at 10 °C, a temperature regime basically ineffective in delaying ripening in MF fruit. This appeared to be the result of an induction of Pp-ACS1 transcription, making this genotype of particular interest for studying temperature stress physiology and ethylene-related ripening processes in peaches. Comparative expression analyses of genes involved in cell wall metabolism pointed out the presence of a negative (Pp-EG4), positive (Pp-endoPG) or no (one member of the PL family) relationship with ethylene at ripening. Results clearly showed that the last stage of firmness decrease (melting) only occurs in fruit producing ethylene and is associated with Pp-endoPG transcript accumulation. The expression of genes involved in ethylene biosynthesis and signalling pathways was evaluated using QRT-PCR. Pp-ACO1 appeared to be induced in SH kept at 10 °C but not at 20 °C. Transient increases in Pp-CTR1 and Pp-EIN2like gene expression have only been detected at the early stages of ripening in samples producing ethylene, indicating that a causal relationship might exist between ethylene and elements of its transduction pathway during peach fruit ripenin

The miRNA-mediated post-transcriptional regulation of maize response to nitrate

Stress responses depend on the correct regulation of gene expression. The discovery that abiotic as well as biotic stresses can regulate miRNA levels, coupled with the identification and functional analyses of stress-associated genes as miRNA targets, provided clues about the vital role that several miRNAs may play in modulating plant resistance to stresses. Nitrogen availability seriously affects crops productivity and environment and the understanding of the miRNA-guided stress regulatory networks should provide new tools for the genetic improvement of nitrogen use efficiency of crops. A recent study revealed the potential role of a number of nitrateresponsive miRNAs in the maize adaptation to nitrate fluctuations. In particular, results obtained suggested that a nitrate depletion might regulate the expression of genes involved in the starvation adaptive response, by affecting the spatiotemporal expression patterns of specific miRNAs