Silvia Farinati

How plants cope with cadmium: staking all on metabolism and gene expression.

Environmental pollution is one of the major problems for human health. Toxic heavy metals are normally present as soil constituents or can also be spread out in the environment by human activity and agricultural techniques. Soil contamination by heavy metals as cadmium, highlights two main aspects: on one side they interfere with the life cycle of plants and therefore reduce crop yields, and on the other hand, once adsorbed and accumulated into the plant tissues, they enter the food chain poisoning animals and humans. Considering this point of view, understanding the mechanism by which plants handle heavy metal exposure, in particular cadmium stress, is a primary goal of plant-biotechnology research or plant breeders whose aim is to create plants that are able to recover high amounts of heavy metals, which can be used for phytoremediation, or identify crop varieties that do not accumulate toxic metal in grains or fruits. In this review we focus on the main symptoms of cadmium toxicity both on root apparatus and shoots. We elucidate the mechanisms that plants activate to prevent absorption or to detoxify toxic metal ions, such as synthesis of phytochelatins, metallothioneins and enzymes involved in stress response. Finally we consider new plant-biotechnology applications that can be applied for phytoremediation.

Regulatory networks of cadmium stress in plants

During their life, plants have to cope with a variety of abiotic stresses. Cadmium is highly toxic to plants, water soluble and therefore promptly adsorbed in tissues and its presence greatly influences the entire plant metabolism. In this review, we focus on the signal pathways responsible for the sensing and transduction of the “metal signal” inside the cell, ultimately driving the activation of transcription factors and consequent expression of genes that enable plants to counteract the heavy metal stress.

Proteomic analysis of Arabidopsis halleri shoots in response to the heavy metals cadmium and zinc and rhizosphere microorganisms.

Arabidopsis halleri has the rare ability to colonize heavy metal‐polluted sites and is an emerging model for research on adaptation and metal hyperaccumulation. The aim of this study was to analyze the effect of plant–microbe interaction on the accumulation of cadmium (Cd) and zinc (Zn) in shoots of an ecotype of A. halleri grown in heavy metal‐contaminated soil and to compare the shoot proteome of plants grown solely in the presence of Cd and Zn or in the presence of these two metals and the autochthonous soil rhizosphere‐derived microorganisms. The results of this analysis emphasized the role of plant–microbe interaction in shoot metal accumulation. Differences in protein expression pattern, identified by a proteomic approach involving 2‐DE and MS, indicated a general upregulation of photosynthesis‐related proteins in plants exposed to metals and to metals plus microorganisms, suggesting that metal accumulation in shoots is an energy‐demanding process. The analysis also showed that proteins involved in plant defense mechanisms were downregulated indicating that heavy metals accumulation in leaves supplies a protection system and highlights a cross‐talk between heavy metal signaling and defense signaling.

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.

The Brassica juncea BjCdR15, an ortholog of Arabidopsis TGA3, is a regulator of cadmium uptake, transport and accumulation in shoots and confers cadmium tolerance in transgenic plants

*A bZIP transcription factor from Brassica juncea (BjCdR15) was isolated by the cDNA-amplified fragment length polymorphism technique after cadmium treatment. Sequence analysis indicated high similarity between BjCdR15 and Arabidopsis TGA3. In Arabidopsis, TGA3 transcription is also induced by cadmium; hence, we investigated whether BjCdR15 is involved in cadmium tolerance and whether it can functionally replace TGA3 protein in Arabidopsis tga3-2 mutant plants. *BjCdR15 expression was detected mainly in the epidermis and vascular system of cadmium-treated plants, and increased in roots and leaves after cadmium treatment. The overexpression of BjCdR15 in Arabidopsis and tobacco enhanced cadmium tolerance: overexpressing plants showed high cadmium accumulation in shoots. Conversely, Arabidopsis tga3-2 mutant plants showed high cadmium content in roots and inhibition of its transport to the shoot. *We demonstrated that BjCdR15 can functionally replace TGA3: in 35S::BjCdR15-tga3-2 plants, the long-distance transport of cadmium from root to shoot was restored and these plants showed an increased cadmium content in shoots compared with all other assays. In addition, BjCdR15/TGA3 regulated the synthesis of phytochelatin synthase and the expression of several metal transporters. *The results indicate that BjCdR15/TGA3 transcription factors play a crucial role in the regulation of cadmium uptake by roots and in its long-distance root to shoot transport. BjCdR15/TGA3 may thus be considered as useful candidates for potential biotechnological applications in the phytoextraction of cadmium from polluted soils.

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 β-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.

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.

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

Agronomically realistic, long-term drought stress mis-regulates some miRNAs and induces the down-regulation of a set of small RNA loci in the maize leaf. 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.

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.