Targeted expression of HvHMA2 increases the mineral content of the inner endosperm in barley.

Cereals are a major source of dietary energy and protein but are nutritionally poor in micronutrients. Zinc (Zn) biofortification of staple crops has been proposed as a promising strategy to combat the global challenge of human Zn-deficiency. The aim of this study was to improve the Zn content in the edible part of the barley (Hordeum vulgare L.) grain by enhancing Zn translocation into the developing seeds. We demonstrate that the barley plasma membrane P-type ATPase Zn transporter, HvHMA2 is an efficient candidate for mineral biofortification of crops. Following a cisgenic approach to produce transgenic homozygous barley line over-expressing HvHMA2 in the transfer cells of the grain, resulted in a doubling of a wide range of nutrients including Zn, iron (Fe), and magnesium (Mg) in the inner endosperm. This article is protected by copyright. All rights reserved.

Identification of Circular RNAs from the Parental Genes Involved in Multiple Aspects of Cellular Metabolism in Barley.

RNA circularization made by head-to-tail back-splicing events is involved in the regulation of gene expression from transcriptional to post-translational levels. By exploiting RNA-Seq data and down-stream analysis, we shed light on the importance of circular RNAs in plants. The results introduce circular RNAs as novel interactors in the regulation of gene expression in plants and imply the comprehensiveness of this regulatory pathway by identifying circular RNAs for a diverse set of genes. These genes are involved in several aspects of cellular metabolism as hormonal signaling, intracellular protein sorting, carbohydrate metabolism and cell-wall biogenesis, respiration, amino acid biosynthesis, transcription and translation, and protein ubiquitination. Additionally, these parental loci of circular RNAs, from both nuclear and mitochondrial genomes, encode for different transcript classes including protein coding transcripts, microRNA, rRNA, and long non-coding/microprotein coding RNAs. The results shed light on the mitochondrial exonic circular RNAs and imply the importance of circular RNAs for regulation of mitochondrial genes. Importantly, we introduce circular RNAs in barley and elucidate their cellular-level alterations across tissues and in response to micronutrients iron and zinc. In further support of circular RNAs' functional roles in plants, we report several cases where fluctuations of circRNAs do not correlate with the levels of their parental-loci encoded linear transcripts.

Deciphering Mineral Homeostasis in Barley Seed Transfer Cells at Transcriptional Level.

In addition to the micronutrient inadequacy of staple crops for optimal human nutrition, a global downtrend in crop-quality has emerged from intensive breeding for yield. This trend will be aggravated by elevated levels of the greenhouse gas carbon dioxide. Therefore, crop biofortification is inevitable to ensure a sustainable supply of minerals to the large part of human population who is dietary dependent on staple crops. This requires a thorough understanding of plant-mineral interactions due to the complexity of mineral homeostasis. Employing RNA sequencing, we here communicate transfer cell specific effects of excess iron and zinc during grain filling in our model crop plant barley. Responding to alterations in mineral contents, we found a long range of different genes and transcripts. Among them, it is worth to highlight the auxin and ethylene signaling factors Arfs, Abcbs, Cand1, Hps4, Hac1, Ecr1, and Ctr1, diurnal fluctuation components Sdg2, Imb1, Lip1, and PhyC, retroelements, sulfur homeostasis components Amp1, Hmt3, Eil3, and Vip1, mineral trafficking components Med16, Cnnm4, Aha2, Clpc1, and Pcbps, and vacuole organization factors Ymr155W, RabG3F, Vps4, and Cbl3. Our analysis introduces new interactors and signifies a broad spectrum of regulatory levels from chromatin remodeling to intracellular protein sorting mechanisms active in the plant mineral homeostasis. The results highlight the importance of storage proteins in metal ion toxicity-resistance and chelation. Interestingly, the protein sorting and recycling factors Exoc7, Cdc1, Sec23A, and Rab11A contributed to the response as well as the polar distributors of metal-transporters ensuring the directional flow of minerals. Alternative isoform switching was found important for plant adaptation and occurred among transcripts coding for identical proteins as well as transcripts coding for protein isoforms. We also identified differences in the alternative-isoform preference between the treatments, indicating metal-affinity shifts among isoforms of metal transporters. Most important, we found the zinc treatment to impair both photosynthesis and respiration. A wide range of transcriptional changes including stress-related genes and negative feedback loops emphasize the importance to withhold mineral contents below certain cellular levels which otherwise might lead to agronomical impeding side-effects. By illustrating new mechanisms, genes, and transcripts, this report provides a solid platform towards understanding the complex network of plant mineral homeostasis.

Correction of gene expression data: Performance-dependency on inter-replicate and inter-treatment biases.

This report investigates for the first time the potential inter-treatment bias source of cell number for gene expression studies. Cell-number bias can affect gene expression analysis when comparing samples with unequal total cellular RNA content or with different RNA extraction efficiencies. For maximal reliability of analysis, therefore, comparisons should be performed at the cellular level. This could be accomplished using an appropriate correction method that can detect and remove the inter-treatment bias for cell-number. Based on inter-treatment variations of reference genes, we introduce an analytical approach to examine the suitability of correction methods by considering the inter-treatment bias as well as the inter-replicate variance, which allows use of the best correction method with minimum residual bias. Analyses of RNA sequencing and microarray data showed that the efficiencies of correction methods are influenced by the inter-treatment bias as well as the inter-replicate variance. Therefore, we recommend inspecting both of the bias sources in order to apply the most efficient correction method. As an alternative correction strategy, sequential application of different correction approaches is also advised.

Dissecting plant iron homeostasis under short and long-term iron fluctuations.

A wealth of information on the different aspects of iron homeostasis in plants has been obtained during the last decade. However, there is no clear road-map integrating the relationships between the various components. The principal aim of the current review is to fill this gap. In this context we discuss the lack of low affinity iron uptake mechanisms in plants, the utilization of a different uptake mechanism by graminaceous plants compared to the others, as well as the roles of riboflavin, ferritin isoforms, nitric oxide, nitrosylation, heme, aconitase, and vacuolar pH. Cross-homeostasis between elements is also considered, with a specific emphasis on the relationship between iron homeostasis and phosphorus and copper deficiencies. As the environment is a crucial parameter for modulating plant responses, we also highlight how diurnal fluctuations govern iron metabolism. Evolutionary aspects of iron homeostasis have so far attracted little attention. Looking into the past can inform us on how long-term oxygen and iron-availability fluctuations have influenced the evolution of iron uptake mechanisms. Finally, we evaluate to what extent this homeostastic road map can be used for the development of novel biofortification strategies in order to alleviate iron deficiency in human.

Coat protein gene sequence analysis of potato virus X and potato virus Y: conserved regions to design gene silencing cassette.

Potato virus X(PVX) and Potato virus Y(PVY) are two of the three most prevalent viruses that cause significant yield declines in potato. Twenty-seven PVX and thirty-seven PVY accessions were analyzed for nucleotide sequence variation of the coat protein gene. The average and variance of genetic distance for PVX were estimated at 0.118 and 0.004 and 0.118 and 0.005 for PVY using the neighbour joining method. Results of phylogenetic trees and their certification via stepwise discriminant analysis led us to classify of PVX sequences in four groups and PVY sequences in three groups. One purpose of this project was to determine suitable conserved regions to make of gene silencing constructs. Length of identified conserved regions were enough to silence of the virus coat protein genes on infected plants, many of which were located consequently with short gap spacers. In this term, some of groups were divided into subgroups to obtain conserved regions under minimum length of25 nt, enough length to design specific diagnostic-primers.