A review of the PIN auxin-efflux transporters, which have important roles in plant development.

The impact of water deficit progression on cytokinin (CK), auxin and abscisic acid (ABA) levels was followed in upper, middle and lower leaves and roots of Nicotiana tabacum L. cv. Wisconsin 38 plants [wild type (WT)]. ABA content was strongly increased during drought stress, especially in upper leaves. In plants with a uniformly elevated total CK content, expressing constitutively the trans-zeatin O-glucosyltransferase gene (35S::ZOG1), a delay in the increase of ABA was observed; later on, ABA levels were comparable with those of WT.As drought progressed, the bioactive CK content in leaves gradually decreased, being maintained longer in the upper leaves of all tested genotypes. Under severe stress (11 d dehydration), a large stimulation of cytokinin oxidase/ dehydrogenase (CKX) activity was monitored in lower leaves, which correlated well with the decrease in bioactive CK levels. This suggests that a gradient of bioactive CKs in favour of upper leaves is established during drought stress, which might be beneficial for the preferential protection of these leaves.During drought, significant accumulation of CKs occurred in roots, partially because of decreased CKX activity. Simultaneously, auxin increased in roots and lower leaves. This indicates that both CKs and auxin play a role in root response to severe drought, which involves the stimulation of primary root growth and branching inhibition.

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Why Be Normal Gene expression profiling by real-time RT-PCR is a much more straight-forward way to quantitate transcript abundance than traditional end point RT-PCR assays. Despite this progress in instrumentation and speed for measuring transcript levels, the need for appropriate normalization remains. Most relative quantification strategies rely on normalization via a reference gene or genes. However, if no suitable housekeeping genes are available, or the use of several internal controls is undesirable, gene expression level may also be normalized to the input RNA amount. Unfortunately, this strategy fails to take into account any variation introduced by the RT step. As an alternative, the RNA-DNA hybrids can be measured after the RT reaction. This approach, however, is limited to experiments that use polyadenylated RNA as input, since with total RNA the fluorescent dye would also bind the double-stranded regions of rRNA. Libus and torchov directly address these limitations with a new measurement approach that uses post-RT RNase digestion and the single-stranded dye RiboGreen. Under these conditions, the fluorescent signal is directly proportional to the amount of single-stranded cDNA, making this method a convenient and quick means of reliable quantitative RT-PCR normalization. - Page 156

Ribosomal protein genes (RPGs) in Saccharomyces cerevisiae are a remarkable regulatory group that may serve as a model for understanding genetic redundancy in evolutionary adaptations. Most RPGs exist as pairs of highly conserved functional paralogs with divergent untranslated regions and introns. We examined the roles of introns in strains with various combinations of intron and gene deletions in RPL22, RPL2, RPL16, RPL37, RPL17, RPS0, and RPS18 paralog pairs. We found that introns inhibited the expression of their genes in the RPL22 pair, with the RPL22B intron conferring a much stronger effect. While the WT RPL22A/RPL22B mRNA ratio was 93/7, the rpl22aΔi/RPL22B and RPL22A/rpl22bΔi ratios were >99/<1 and 60/40, respectively. The intron in RPL2A stimulated the expression of its own gene, but the removal of the other introns had little effect on expression of the corresponding gene pair. Rpl22 protein abundances corresponded to changes in mRNAs.Using splicing reporters containing endogenous intron sequences, we demonstrated that these effects were due to the inhibition of splicing by Rpl22 proteins but not by their RNA-binding mutant versions. Indeed, only WT Rpl22A/Rpl22B proteins (but not the mutants) interacted in a yeast three-hybrid system with an RPL22B intronic region between bp 165 and 236. Transcriptome analysis showed that both the total level of Rpl22 and the A/B ratio were important for maintaining the WT phenotype. The data presented here support the contention that the Rpl22B protein has a paralog-specific role.The RPL22 singleton of Kluyveromyces lactis, which did not undergo whole genome duplication, also responded to Rpl22-mediated inhibition in K. lactis cells. Vice versa, the overproduction of the K. lactis protein reduced the expression of RPL22A/B in S. cerevisiae. The extraribosomal function of of the K. lactis Rpl22 suggests that the loop regulating RPL22 paralogs of S. cerevisiae evolved from autoregulation.

Pre-mRNA splicing represents an important regulatory layer of eukaryotic gene expression. In the simple budding yeast Saccharomyces cerevisiae, about one-third of all mRNA molecules undergo splicing, and splicing efficiency is tightly regulated, for example, during meiotic differentiation. S. cerevisiae features a streamlined, evolutionarily highly conserved splicing machinery and serves as a favourite model for studies of various aspects of splicing. RNA-seq represents a robust, versatile, and affordable technique for transcriptome interrogation, which can also be used to study splicing efficiency. However, convenient bioinformatics tools for the analysis of splicing efficiency from yeast RNA-seq data are lacking. We present a complete workflow for the calculation of genome-wide splicing efficiency in S. cerevisiae using strand-specific RNA-seq data. Our pipeline takes sequencing reads in the FASTQ format and provides splicing efficiency values for the 5′ and 3′ splice junctions of each intron. The pipeline is based on up-to-date open-source software tools and requires very limited input from the user. We provide all relevant scripts in a ready-to-use form. We demonstrate the functionality of the workflow using RNA-seq datasets from three spliceosome mutants. The workflow should prove useful for studies of yeast splicing mutants or of regulated splicing, for example, under specific growth conditions.