Not Only Editing: A Cas-Cade of CRISPR/Cas-Based Tools for Functional Genomics in Plants and Animals.

The advent of CRISPR/Cas9 technology has revolutionized genome editing, enabling the attainment of once-unimaginable goals. CRISPR/Cas's groundbreaking attributes lie in its simplicity, versatility, universality, and independence from customized DNA-protein systems, erasing the need for specialized expertise and broadening its scope of applications. It is therefore more and more used for genome modification including the generation of mutants. Beyond such editing scopes, the recent development of novel or modified Cas-based systems has spawned an array of additional biotechnological tools, empowering both fundamental and applied research. Precisely targeting DNA or RNA sequences, the CRISPR/Cas system has been harnessed in fields as diverse as gene regulation, deepening insights into gene expression, epigenetic changes, genome spatial organization, and chromatin dynamics. Furthermore, it aids in genome imaging and sequencing, as well as effective identification and countering of viral pathogens in plants and animals. All in all, the non-editing aspect of CRISPR/Cas exhibits tremendous potential across diverse domains, including diagnostics, biotechnology, and fundamental research. This article reviews and critically evaluates the primary CRISPR/Cas-based tools developed for plants and animals, underlining their transformative impact.

Current insights and advances into plant male sterility: new precision breeding technology based on genome editing applications.

Plant male sterility (MS) represents the inability of the plant to generate functional anthers, pollen, or male gametes. Developing MS lines represents one of the most important challenges in plant breeding programs, since the establishment of MS lines is a major goal in F1 hybrid production. For these reasons, MS lines have been developed in several species of economic interest, particularly in horticultural crops and ornamental plants. Over the years, MS has been accomplished through many different techniques ranging from approaches based on cross-mediated conventional breeding methods, to advanced devices based on knowledge of genetics and genomics to the most advanced molecular technologies based on genome editing (GE). GE methods, in particular gene knockout mediated by CRISPR/Cas-related tools, have resulted in flexible and successful strategic ideas used to alter the function of key genes, regulating numerous biological processes including MS. These precision breeding technologies are less time-consuming and can accelerate the creation of new genetic variability with the accumulation of favorable alleles, able to dramatically change the biological process and resulting in a potential efficiency of cultivar development bypassing sexual crosses. The main goal of this manuscript is to provide a general overview of insights and advances into plant male sterility, focusing the attention on the recent new breeding GE-based applications capable of inducing MS by targeting specific nuclear genic loci. A summary of the mechanisms underlying the recent CRISPR technology and relative success applications are described for the main crop and ornamental species. The future challenges and new potential applications of CRISPR/Cas systems in MS mutant production and other potential opportunities will be discussed, as generating CRISPR-edited DNA-free by transient transformation system and transgenerational gene editing for introducing desirable alleles and for precision breeding strategies.

MIK2 is a candidate gene of the S-locus for sporophytic self-incompatibility in chicory (Cichorium intybus, Asteraceae).

The Cichorium genus offers a unique opportunity to study the sporophytic self-incompatibility (SSI) system, being composed of species characterized by highly efficient self-incompatibility (e.g., C. intybus) and complete self-compatibility (e.g., C. endivia). To this end, the chicory genome was used to map seven previously identified SSI locus-associated markers. The region containing the S-locus was therefore restricted to an ~4 M bp window on chromosome 5. Among the genes predicted in this region, MDIS1 INTERACTING RECEPTOR LIKE KINASE 2 (ciMIK2) was particularly promising as a candidate for SSI. Its ortholog in Arabidopsis (atMIK2) is involved in pollen-stigma recognition reactions, and its protein structure is similar to that of S-receptor kinase (SRK), a key component of the SSI system in the Brassica genus. The amplification and sequencing of MIK2 in chicory and endive accessions revealed two contrasting scenarios. In C. endivia, MIK2 was fully conserved even when comparing different botanical varieties (i.e., smooth and curly endive). In C. intybus, 387 polymorphic positions and 3 INDELs were identified when comparing accessions of different biotypes all belonging to the same botanical variety (i.e., radicchio). The polymorphism distribution throughout the gene was uneven, with hypervariable domains preferentially localized in the LRR-rich extracellular region, putatively identified as the receptor domain. The gene was hypothesized to be under positive selection, as the nonsynonymous mutations were more than double the synonymous ones (dN/dS = 2.17). An analogous situation was observed when analyzing the first 500 bp of the MIK2 promoter: no SNPs were observed among the endive samples, whereas 44 SNPs and 6 INDELs were detected among the chicory samples. Further analyses are needed to confirm the role of MIK2 in SSI and to demonstrate whether the 23 species-specific nonsynonymous SNPs in the CDS and/or the species-specific 10 bp-INDEL found in a CCAAT box region of the promoter are responsible for the contrasting sexual behaviors of chicory and endive.

Dissecting the effect of soil on plant phenology and berry transcriptional plasticity in two Italian grapevine varieties (Vitis vinifera L.).

Grapevine embodies a fascinating species as regards phenotypic plasticity and genotype-per-environment interactions. The terroir, namely the set of agri-environmental factors to which a variety is subjected, can influence the phenotype at the physiological, molecular, and biochemical level, representing an important phenomenon connected to the typicality of productions. We investigated the determinants of plasticity by conducting a field-experiment where all terroir variables, except soil, were kept as constant as possible. We isolated the effect of soils collected from different areas, on phenology, physiology, and transcriptional responses of skin and flesh of a red and a white variety of great economic value: Corvina and Glera. Molecular results, together with physio-phenological parameters, suggest a specific effect of soil on grapevine plastic response, highlighting a higher transcriptional plasticity of Glera in respect to Corvina and a marked response of skin compared to flesh. Using a novel statistical approach, we identified clusters of plastic genes subjected to the specific influence of soil. These findings could represent an issue of applicative value, posing the basis for targeted agricultural practices to enhance the desired characteristics for any soil/cultivar combination, to improve vineyards management for a better resource usage and to valorize vineyards uniqueness maximizing the terroir-effect.

Pipeline to Design Inbred Lines and F1 Hybrids of Leaf Chicory (Radicchio) Using Male Sterility and Genotyping-by-Sequencing.

Chicory, a horticultural crop cultivated worldwide, presents many botanical varieties and local biotypes. Among these, cultivars of the Italian radicchio group of the pure species Cichorium intybus L. and its interspecific hybrids with Cichorium endivia L.-as the "Red of Chioggia" biotype-includes several phenotypes. This study uses a pipeline to address the marker-assisted breeding of F1 hybrids: it presents the genotyping-by-sequencing results of four elite inbred lines using a RADseq approach and an original molecular assay based on CAPS markers for screening mutants with nuclear male sterility in the radicchio of Chioggia. A total of 2953 SNP-carrying RADtags were identified and used to compute the actual estimates of homozygosity and overall genetic similarity and uniformity of the populations, as well as to determine their genetic distinctiveness and differentiation. Molecular data were further used to investigate the genomic distribution of the RADtags among the two Cichorium species, allowing their mapping in 1131 and 1071 coding sequences in chicory and endive, respectively. Paralleling this, an assay to screen the genotype at the male sterility locus Cims-1 was developed to discriminate wild-type and mutant alleles of the causative gene myb80-like. Moreover, a RADtag mapped close to this genomic region proved the potential application of this method for future marker-assisted selection tools. Finally, after combining the genotype information of the core collection, the best 10 individuals from each inbred line were selected to compute the observed genetic similarity as a measure of uniformity as well as the expected homozygosity and heterozygosity estimates scorable by the putative progenies derived from selfing (pollen parent) and full-sibling (seed parent) or pair-wise crossing (F1 hybrids). This predictive approach was conducted as a pilot study to understand the potential application of RADseq in the fine tuning of molecular marker-assisted breeding strategies aimed at the development of inbred lines and F1 hybrids in leaf chicory.

Can allele-specific loop-mediated isothermal amplification be used for rapid detection of target-site herbicide resistance in Lolium spp.?

BACKGROUND: Herbicide resistance is one of the threats to modern agriculture and its early detection is one of the most effective components for sustainable resistance management strategies. Many techniques have been used for target-site-resistance detection. Allele-Specific Loop-Mediated Isothermal Amplification (AS-LAMP) was evaluated as a possible rapid diagnostic method for acetyl-CoA carboxylase (ACCase) and acetolactate synthase (ALS) inhibiting herbicides resistance in Lolium spp. RESULTS: AS-LAMP protocols were set up for the most frequent mutations responsible for herbicide resistance to ALS (positions 197, 376 and 574) and ACCase (positions 1781, 2041 and 2078) inhibitors in previously characterized and genotyped Lolium spp. POPULATIONS: A validation step on new putative resistant populations gave the overview of a possible use of this tool for herbicide resistance diagnosis in Lolium spp. Regarding the ACCase inhibitor pinoxaden, in more than 65% of the analysed plants, the LAMP assay and genotyping were in keeping, whereas the results were not consistent when ALS inhibitors resistance was considered. Limitations on the use of this technique for herbicide resistance detection in the allogamous Lolium spp. are discussed. CONCLUSIONS: The LAMP method used for the detection of target-site resistance in weed species could be applicable with target genes that do not have high genetic variability, such as ACCase gene in Lolium spp.

Past, present, and future of genetic strategies to control tolerance to the main fungal and oomycete pathogens of grapevine.

The production of high-quality wines is strictly related to the correct management of the vineyard, which guarantees good yields and grapes with the right characteristics required for subsequent vinification. Winegrowers face a variety of challenges during the grapevine cultivation cycle: the most notorious are fungal and oomycete diseases such as downy mildew, powdery mildew, and gray mold. If not properly addressed, these diseases can irremediably compromise the harvest, with disastrous consequences for the production and wine economy. Conventional defense methods used in the past involved chemical pesticides. However, such approaches are in conflict with the growing attention to environmental sustainability and shifts from the uncontrolled use of chemicals to the use of integrated approaches for crop protection. Improvements in genetic knowledge and the availability of novel biotechnologies have created new scenarios for possibly producing grapes with a reduced, if not almost zero, impact. Here, the main approaches used to protect grapevines from fungal and oomycete diseases are reviewed, starting from conventional breeding, which allowed the establishment of new resistant varieties, followed by biotechnological methods, such as transgenesis, cisgenesis, intragenesis, and genome editing, and ending with more recent perspectives concerning the application of new products based on RNAi technology. Evidence of their effectiveness, as well as potential risks and limitations based on the current legislative situation, are critically discussed.

An efficient chromatin immunoprecipitation (ChIP) protocol for studying histone modifications in peach reproductive tissues.

BACKGROUND: Perennial fruit trees display a growth behaviour characterized by annual cycling between growth and dormancy, with complex physiological features. Rosaceae fruit trees represent excellent models for studying not only the fruit growth/patterning but also the progression of the reproductive cycle depending upon the impact of climate conditions. Additionally, current developments in high-throughput technologies have impacted Rosaceae tree research while investigating genome structure and function as well as (epi)genetic mechanisms involved in important developmental and environmental response processes during fruit tree growth. Among epigenetic mechanisms, chromatin remodelling mediated by histone modifications and other chromatin-related processes play a crucial role in gene modulation, controlling gene expression. Chromatin immunoprecipitation is an effective technique to investigate chromatin dynamics in plants. This technique is generally applied for studies on chromatin states and enrichment of post-transcriptional modifications (PTMs) in histone proteins. RESULTS: Peach is considered a model organism among climacteric fruits in the Rosaceae family for studies on bud formation, dormancy, and organ differentiation. In our work, we have primarily established specific protocols for chromatin extraction and immunoprecipitation in reproductive tissues of peach (Prunus persica). Subsequently, we focused our investigations on the role of two chromatin marks, namely the trimethylation of histone H3 at lysine in position 4 (H3K4me3) and trimethylation of histone H3 at lysine 27 (H3K27me3) in modulating specific gene expression. Bud dormancy and fruit growth were investigated in a nectarine genotype called Fantasia as our model system. CONCLUSIONS: We present general strategies to optimize ChIP protocols for buds and mesocarp tissues of peach and analyze the correlation between gene expression and chromatin mark enrichment/depletion. The procedures proposed may be useful to evaluate any involvement of histone modifications in the regulation of gene expression during bud dormancy progression and core ripening in fruits.

Regulation of Fruit Growth in a Peach Slow Ripening Phenotype.

Consumers' choices are mainly based on fruit external characteristics such as the final size, weight, and shape. The majority of edible fruit are by tree fruit species, among which peach is the genomic and genetic reference for Prunus. In this research, we used a peach with a slow ripening (SR) phenotype, identified in the Fantasia (FAN) nectarine, associated with misregulation of genes involved in mesocarp identity and showing a reduction of final fruit size. By investigating the ploidy level, we observed a progressive increase in endoreduplication in mesocarp, which occurred in the late phases of FAN fruit development, but not in SR fruit. During fruit growth, we also detected that genes involved in endoreduplication were differentially modulated in FAN compared to SR. The differential transcriptional outputs were consistent with different chromatin states at loci of endoreduplication genes. The impaired expression of genes controlling cell cycle and endocycle as well as those claimed to play a role in fruit tissue identity result in the small final size of SR fruit.

Epigenetic signatures of stress adaptation and flowering regulation in response to extended drought and recovery in Zea mays.

During their lifespan, plants respond to a multitude of stressful factors. Dynamic changes in chromatin and concomitant transcriptional variations control stress response and adaptation, with epigenetic memory mechanisms integrating environmental conditions and appropriate developmental programs over the time. Here we analyzed transcriptome and genome-wide histone modifications of maize plants subjected to a mild and prolonged drought stress just before the flowering transition. Stress was followed by a complete recovery period to evaluate drought memory mechanisms. Three categories of stress-memory genes were identified: i) "transcriptional memory" genes, with stable transcriptional changes persisting after the recovery; ii) "epigenetic memory candidate" genes in which stress-induced chromatin changes persist longer than the stimulus, in absence of transcriptional changes; iii) "delayed memory" genes, not immediately affected by the stress, but perceiving and storing stress signal for a delayed response. This last memory mechanism is described for the first time in drought response. In addition, applied drought stress altered floral patterning, possibly by affecting expression and chromatin of flowering regulatory genes. Altogether, we provided a genome-wide map of the coordination between genes and chromatin marks utilized by plants to adapt to a stressful environment, describing how this serves as a backbone for setting stress memory.

Control of Maize Vegetative and Reproductive Development, Fertility, and rRNAs Silencing by HISTONE DEACETYLASE 108.

Histone deacetylases (HDACs) catalyze the removal of acetyl groups from acetylated histone tails that consequently interact more closely with DNA, leading to chromatin state refractory to transcription. Zea mays HDA108 belongs to the Rpd3/HDA1 HDAC family and is ubiquitously expressed during development. The newly isolated hda108/hda108 insertional mutant exhibited many developmental defects: significant reduction in plant height, alterations of shoot and leaf development, and alterations of inflorescence patterning and fertility. Western blot analyses and immunolocalization experiments revealed an evident increase in histone acetylation, accompanied by a marked reduction in H3K9 dimethylation, in mutant nuclei. The DNA methylation status, in the CHG sequence context, and the transcript level of ribosomal sequences were also affected in hda108 mutants, while enrichment in H3 and H4 acetylation characterizes both repetitive and nonrepetitive transcriptional up-regulated loci. RNA-Seq of both young leaf and anthers indicated that transcription factor expression is highly affected and that the pollen developmental program is disrupted in hda108 mutants. Crosses between hda108/hda108 and epiregulator mutants did not produce any double mutant progeny indicating possible genetic interactions of HDA108 with distinct epigenetic pathways. Our findings indicate that HDA108 is directly involved in regulation of maize development, fertility, and epigenetic regulation of genome activity.

De Novo Identification of sRNA Loci and Non-coding RNAs by High-Throughput Sequencing.

Non-coding RNA transcripts, such as long non-coding RNAs, miRNAs, siRNAs, and transposon-originating transcripts, are involved in the regulation of RNA stability, protein translation, and/or the modulation of chromatin states. RNA-Seq can be used to catalog this diversity of novel transcripts and a joint analysis of these transcriptomic data can provide useful insights into epigenetic regulation of dynamic responses such as the stress response, which may not be deciphered from individual analysis of single transcript categories. Here, we present a protocol that allows the identification and analysis of small RNAs and long non-coding RNAs, together with the comparison of these species between different sample types.

Maize RNA PolIV affects the expression of genes with nearby TE insertions and has a genome-wide repressive impact on transcription.

BACKGROUND: RNA-directed DNA methylation (RdDM) is a plant-specific epigenetic process that relies on the RNA polymerase IV (Pol IV) for the production of 24 nucleotide small interfering RNAs (siRNA) that guide the cytosine methylation and silencing of genes and transposons. Zea mays RPD1/RMR6 gene encodes the largest subunit of Pol IV and is required for normal plant development, paramutation, transcriptional repression of certain transposable elements (TEs) and transcriptional regulation of specific alleles. RESULTS: In this study we applied a total RNA-Seq approach to compare the B73 and rpd1/rmr6 leaf transcriptomes. Although previous studies indicated that loss of siRNAs production in RdDM mutants provokes a strong loss of CHH DNA methylation but not massive gene or TEs transcriptional activation in both Arabidopsis and maize, our total RNA-Seq analysis of rpd1/rmr6 transcriptome reveals that loss of Pol IV activity causes a global increase in the transcribed fraction of the maize genome. Our results point to the genes with nearby TE insertions as being the most strongly affected by Pol IV-mediated gene silencing. TEs modulation of nearby gene expression is linked to alternative methylation profiles on gene flanking regions, and these profiles are strictly dependent on specific characteristics of the TE member inserted. Although Pol IV is essential for the biogenesis of siRNAs, the genes with associated siRNA loci are less affected by the pol IV mutation. CONCLUSIONS: This deep and integrated analysis of gene expression, TEs distribution, smallRNA targeting and DNA methylation levels, reveals that loss of Pol IV activity globally affects genome regulation, pointing at TEs as modulator of nearby gene expression and indicating the existence of multiple level epigenetic silencing mechanisms. Our results also suggest a predominant role of the Pol IV-mediated RdDM pathway in genome dominance regulation, and subgenome stability and evolution in maize.

Rosaceae Fruit Development, Ripening and Post-harvest: An Epigenetic Perspective.

Rosaceae is a family with an extraordinary spectrum of fruit types, including fleshy peach, apple, and strawberry that provide unique contributions to a healthy diet for consumers, and represent an excellent model for studying fruit patterning and development. In recent years, many efforts have been made to unravel regulatory mechanism underlying the hormonal, transcriptomic, proteomic and metabolomic changes occurring during Rosaceae fruit development. More recently, several studies on fleshy (tomato) and dry (Arabidopsis) fruit model have contributed to a better understanding of epigenetic mechanisms underlying important heritable crop traits, such as ripening and stress response. In this context and summing up the results obtained so far, this review aims to collect the available information on epigenetic mechanisms that may provide an additional level in gene transcription regulation, thus influencing and driving the entire Rosaceae fruit developmental process. The whole body of information suggests that Rosaceae fruit could become also a model for studying the epigenetic basis of economically important phenotypes, allowing for their more efficient exploitation in plant breeding.

Stress-induced and epigenetic-mediated maize transcriptome regulation study by means of transcriptome reannotation and differential expression analysis.

Plant's response and adaptation to abiotic stresses involve sophisticated genetic and epigenetic regulatory systems. To obtain a global view of molecular response to osmotic stresses, including the non-coding portion of genome, we conducted a total leaf transcriptome analysis on maize plants subjected to prolonged drought and salt stresses. Stress application to both B73 wild type and the epiregulator mutant rpd1-1/rmr6 allowed dissection of the epigenetic component of stress response. Coupling total RNA-Seq and transcriptome re-assembly we annotated thousands of new maize transcripts, together with 13,387 lncRNAs that may play critical roles in regulating gene expression. Differential expression analysis revealed hundreds of genes modulated by long-term stress application, including also many lncRNAs and transposons specifically induced by stresses. The amplitude and dynamic of the stress-modulated gene sets are very different between B73 and rpd1-1/rmr6 mutant plants, as result of stress-like effect on genome regulation caused by the mutation itself, which activates many stress-related genes even in control condition. The analyzed extensive set of total RNA-Seq data, together with the improvement of the transcriptome and the identification of the non-coding portion of the transcriptome give a revealing insight into the genetic and epigenetic mechanism responsible for maize molecular response to abiotic stresses.

Genome-Wide Characterization of Maize Small RNA Loci and Their Regulation in the required to maintain repression6-1 (rmr6-1) Mutant and Long-Term Abiotic Stresses.

Endogenous small RNAs (sRNAs) contribute to gene regulation and genome homeostasis, but their activities and functions are incompletely known. The maize genome has a high number of transposable elements (TEs; almost 85%), some of which spawn abundant sRNAs. We performed sRNA and total RNA sequencing from control and abiotically stressed B73 wild-type plants and rmr6-1 mutants. RMR6 encodes the largest subunit of the RNA polymerase IV complex and is responsible for accumulation of most 24-nucleotide (nt) small interfering RNAs (siRNAs). We identified novel MIRNA loci and verified miR399 target conservation in maize. RMR6-dependent 23-24 nt siRNA loci were specifically enriched in the upstream region of the most highly expressed genes. Most genes misregulated in rmr6-1 did not show a significant correlation with loss of flanking siRNAs, but we identified one gene supporting existing models of direct gene regulation by TE-derived siRNAs. Long-term drought correlated with changes of miRNA and sRNA accumulation, in particular inducing down-regulation of a set of sRNA loci in the wild-typeleaf.

Time course of biochemical, physiological, and molecular responses to field-mimicked conditions of drought, salinity, and recovery in two maize lines.

Drought and salinity stresses will have a high impact on future crop productivity, due to climate change and the increased competition for land, water, and energy. The response to drought (WS), salinity (SS), and the combined stresses (WS+SS) was monitored in two maize lines: the inbred B73 and an F1 commercial stress-tolerant hybrid. A protocol mimicking field progressive stress conditions was developed and its effect on plant growth analyzed at different time points. The results indicated that the stresses limited growth in the hybrid and arrested it in the inbred line. In SS, the two genotypes had different ion accumulation and translocation capacity, particularly for Na(+) and Cl(-). Moreover, the hybrid perceived the stress, reduced all the analyzed physiological parameters, and kept them reduced until the recovery. B73 decreased all physiological parameters more gradually, being affected mainly by SS. Both lines recovered better from WS than the other stresses. Molecular analysis revealed a diverse modulation of some stress markers in the two genotypes, reflecting their different response to stresses. Combining biochemical and physiological data with expression analyses yielded insight into the mechanisms regulating the different stress tolerance of the two lines.

Pseudomonas putida response to cadmium: changes in membrane and cytosolic proteomes.

Pseudomonas putida is a saprophytic bacterium with remarkable environmental adaptability and the capacity to tolerate high concentrations of heavy metals. The strain P. putida-Cd001 was isolated from soil contaminated with Cd, Zn and Pb. Membrane-associated and cytosolic proteomes were analyzed to identify proteins whose expression was modulated in response to 250 µM CdSO4. We identified 44 protein spots in the membrane and 21 in the cytosolic fraction differentially expressed in Cd-treated samples compared to untreated controls. Outer membrane porins from the OprD and OprI families were less abundant in bacteria exposed to Cd, whereas those from the OprF and OprL, OprH and OprB families were more abundant, reflecting the increased need to acquire energy sources, the need to maintain membrane integrity and the process of adaptation. Components of the efflux system, such as the CzcB subunit of the CBA system, were also induced by Cd. Analysis of the cytosolic proteome revealed that proteins involved in protein synthesis, degradation and folding were induced along with enzymes that combat oxidative stress, showing that the entire bacterial proteome is modulated by heavy metal exposure. This analysis provides new insights into the adaptation mechanisms used by P. putida-Cd001 to survive in Cd-polluted environments.

The Maize PIN Gene Family of Auxin Transporters.

Auxin is a key regulator of plant development and its differential distribution in plant tissues, established by a polar cell to cell transport, can trigger a wide range of developmental processes. A few members of the two families of auxin efflux transport proteins, PIN-formed (PIN) and P-glycoprotein (ABCB/PGP), have so far been characterized in maize. Nine new Zea mays auxin efflux carriers PIN family members and two maize PIN-like genes have now been identified. Four members of PIN1 (named ZmPIN1a-d) cluster, one gene homologous to AtPIN2 (ZmPIN2), three orthologs of PIN5 (ZmPIN5a-c), one gene paired with AtPIN8 (ZmPIN8), and three monocot-specific PINs (ZmPIN9, ZmPIN10a, and ZmPIN10b) were cloned and the phylogenetic relationships between early-land plants, monocots, and eudicots PIN proteins investigated, including the new maize PIN proteins. Tissue-specific expression patterns of the 12 maize PIN genes, 2 PIN-like genes and ZmABCB1, an ABCB auxin efflux carrier, were analyzed together with protein localization and auxin accumulation patterns in normal conditions and in response to drug applications. ZmPIN gene transcripts have overlapping expression domains in the root apex, during male and female inflorescence differentiation and kernel development. However, some PIN family members have specific tissue localization: ZmPIN1d transcript marks the L1 layer of the shoot apical meristem and inflorescence meristem during the flowering transition and the monocot-specific ZmPIN9 is expressed in the root endodermis and pericycle. The phylogenetic and gene structure analyses together with the expression pattern of the ZmPIN gene family indicate that subfunctionalization of some maize PINs can be associated to the differentiation and development of monocot-specific organs and tissues and might have occurred after the divergence between dicots and monocots.

Interaction between selected bacterial strains and Arabidopsis halleri modulates shoot proteome and cadmium and zinc accumulation.

The effects of plant-microbe interactions between the hyperaccumulator Arabidopsis halleri and eight bacterial strains, isolated from the rhizosphere of A. halleri plants grown in a cadmium- and zinc-contaminated site, were analysed for shoot metal accumulation, shoot proteome, and the transcription of genes involved in plant metal homeostasis and hyperaccumulation. Cadmium and zinc concentrations were lower in the shoots of plants cultivated in the presence of these metals plus the selected bacterial strains compared with plants grown solely with these metals or, as previously reported, with plants grown with these metals plus the autochthonous rhizosphere-derived microorganisms. The shoot proteome of plants cultivated in the presence of these selected bacterial strains plus metals, showed an increased abundance of photosynthesis- and abiotic stress-related proteins (e.g. subunits of the photosynthetic complexes, Rubisco, superoxide dismutase, and malate dehydrogenase) counteracted by a decreased amount of plant defence-related proteins (e.g. endochitinases, vegetative storage proteins, and ß-glucosidase). The transcription of several homeostasis genes was modulated by the microbial communities and by Cd and Zn content in the shoot. Altogether these results highlight the importance of plant-microbe interactions in plant protein expression and metal accumulation and emphasize the possibility of exploiting microbial consortia for increasing or decreasing shoot metal content.