Heat Stress Affects Pi-related Genes Expression and Inorganic Phosphate Deposition/Accumulation in Barley

Phosphorus (P) in plants is taken from soil as an inorganic phosphate (Pi) and is one of the most important macroelements in growth and development. Plants actively react to Pi starvation by the induced expression of Pi transporters, MIR399, MIR827, and miR399 molecular sponge – IPS1 genes and by the decreased expression of the ubiquitin-conjugating enzyme E2 (PHOSPHATE2 – PHO2) and Pi sensing and transport SPX-MFS genes. The PHO2 protein is involved in the degradation of Pi transporters PHT1;1 (from soil to roots) and PHO1 (from roots to shoots). The decreased expression of PHO2 leads to Pi accumulation in shoots. In contrast, the pho1 mutant shows a decreased level of Pi concentration in shoots. Finally, Pi starvation leads to decreased Pi concentration in all plant tissues. Little is known about plant Pi homeostasis in other abiotic stress conditions. We found that, during the first hour of heat stress, Pi accumulated in barley shoots but not in the roots, and transcriptomic data analysis as well as RT-qPCR led us to propose an explanation for this phenomenon. Pi transport inhibition from soil to roots is balanced by lower Pi efflux from roots to shoots directed by the PHO1 transporter. In shoots, the PHO2 mRNA level is decreased, leading to an increased Pi level. We concluded that Pi homeostasis in barley during heat stress is maintained by dynamic changes in Pi-related genes expression

Arabidopsis thaliana microRNA162 level is posttranscriptionally regulated via splicing and polyadenylation site selection

Arabidopsis microRNA162 (miRNA162) level regulation was studied under abiotic stresses, such as drought and salinity. The TaqMan® microRNA assay proved that A. thaliana miRNA162 level was elevated under these stresses, confirming its salt and drought responsiveness. The promoter region analyses of A. thaliana miRNA162a and b genes (MIR162a and MIR162b) identified numerous salinity and drought responsive elements. However, our results indicated that Arabidopsis MIR162a was presumably the main locus responsible for the mature ath-miRNA162 accumulation under the stresses tested, and the MIR162b was generally rather weakly expressed, both in control and under the stress conditions. The MIR162a structure was confirmed to be complex and the pri-miRNA162a hairpin structure was shown to span an alternative exon and an intron. The MIR162a transcription generated a few pri-miRNA162a splicing isoforms that could be functional and non-functional. Upon drought and salinity stresses, the regulation of the pri-miRNA162a alternative splicing pattern revealed an increase of a functional pri-miR162a isoform and a preferential distal polyA site selection under the stress conditions. Apart from the potential transcriptional regulation of the miRNA genes (MIRs) expression, the data obtained point to an essential role of posttranscriptional regulation of Arabidopsis microRNA162 level

Active 5′ splice sites regulate the biogenesis efficiency of Arabidopsis microRNAs derived from intron-containing genes

Arabidopsis, miR402 that is encoded within the first intron of a protein-coding gene At1g77230, is induced by heat stress. Its upregulation correlates with splicing inhibition and intronic proximal polyA site selection. It suggests that miR402 is not processed from an intron, but rather from a shorter transcript after selection of the proximal polyA site within this intron. Recently, introns and active 5′ splice sites (5′ss’) have been shown to stimulate the accumulation of miRNAs encoded within the first exons of intron-containing MIR genes. In contrast, we have observed the opposite effect of splicing inhibition on intronic miR402 production. Transient expression experiments performed in tobacco leaves revealed a significant accumulation of the intronic mature miR402 when the 5′ss of the miR402-hosting intron was inactivated. In contrast, when the miR402 stem-loop structure was moved into the first exon, mutation of the first-intron 5′ss resulted in a decrease in the miRNA level. Thus, the 5′ss controls the efficiency of miRNA biogenesis. We also show that the SERRATE protein (a key component of the plant microprocessor) colocalizes and interacts with several U1 snRNP auxiliary proteins. We postulate that SERRATE-spliceosome connections have a direct effect on miRNA maturation

Virus-Induced Gene Silencing-Based Functional Characterization of Genes Associated with Powdery Mildew Resistance in Barley

We successfully implemented virus-induced gene silencing (VIGS) in barley (Hordeum vulgare) for the functional characterization of genes required for Mla13-mediated resistance toward the biotrophic barley pathogen Blumeria graminis f. sp. hordei. Initially, barley cultivars were screened for their ability to host the barley stripe mosaic virus (BSMV)-VIGS vector by allowing its replication and systemic movement without causing excessive symptoms. Phytoene desaturase silencing leading to photobleaching was used as a phenotypic marker alongside reverse transcription-PCR data to characterize the silencing response at the molecular level. Barley cultivar Clansman, harboring the Mla13 resistance gene, was chosen as the most suitable host for BSMV-VIGS-based functional characterization of Rar1, Sgt1, and Hsp90 in the Mla-mediated resistance toward powdery mildew. BSMV-induced gene silencing of these candidate genes, which are associated in many but not all race-specific pathways, proved to be robust and could be detected at both mRNA and protein levels for up to 21 d postinoculation. Systemic silencing was observed not only in the newly developed leaves from the main stem but also in axillary shoots. By examining fungal development from an incompatible mildew strain carrying the cognate Avr13 gene on plants BSMV silenced for Rar1, Sgt1, and Hsp90, a resistance-breaking phenotype was observed, while plants infected with BSMV control constructs remained resistant. We demonstrate that Hsp90 is a required component for Mla13-mediated race-specific resistance and that BSMV-induced VIGS is a powerful tool to characterize genes involved in pathogen resistance in barley

Determinants of plant U12-dependent intron splicing efficiency

Factors affecting splicing of plant U12-dependent introns have been examined by extensive mutational analyses in an in vivo tobacco (Nicotiana tabacum) protoplast system using introns from three different Arabidopsis thaliana genes: CBP20, GSH2, and LD. The results provide evidence that splicing efficiency of plant U12 introns depends on a combination of factors, including UA content, exon bridging interactions between the U12 intron and flanking U2-dependent introns, and exon splicing enhancer sequences (ESEs). Unexpectedly, all three plant U12 introns required an adenosine at the upstream purine position in the branchpoint consensus UCCUURAUY. The exon upstream of the LD U12 intron is a major determinant of its higher level of splicing efficiency and potentially contains two ESE regions. These results suggest that in plants, U12 introns represent a level at which expression of their host genes can be regulated