Annotation and profiling of barley <i>GLYCOGEN SYNTHASE3/Shaggy</i>-like genes indicated shift in organ-preferential expression

<div><p>GLYCOGEN SYNTHASE KINASE3/Shaggy-like kinases (GSKs) represent a highly conserved group of proteins found in all eukaryotes. In plants they are encoded by multigene families and integrate signaling of brassinosteroids, auxin and abscisic acid in wide range of physiological and developmental processes with a strong impact on plant responses to environmental and biotic factors. Based on comprehensively studied structures of 10 <i>Arabidopsis thaliana GSK</i> genes and encoded proteins we report identification and phylogenetic reconstruction of 7 transcriptionally active <i>GSK</i> genes in barley. We re-evaluated annotation of the <i>GSK</i> genes in the current barley genome (Hv_IBSC_PGSB_v2) and provided data that a single gene annotated in the previous barley genome ensemble should be retained in the current one. The novel structure of another <i>GSK</i>, predicted in Hv_IBSC_PGSB_v2 to encode both GSK and amine oxidase domains, was proposed and experimentally confirmed based on the syntenic region in <i>Brachypodium distachyon</i>. The genes were assigned to 4 groups based on their encoded amino acid sequences and protein kinase domains. The analysis confirmed high level of conservation of functional protein domains and motifs among plant GSKs and the identified barley orthologs. Each of the seven identified <i>HvGSK</i> genes was expressed indicating semi-constitutive regulation in all tested organs and developmental stages. Regulation patterns of <i>GSKs</i> from the indicated groups showed a shift in organ-preferential expression in <i>A</i>. <i>thaliana</i> and barley illustrating diversification of biological roles of individual <i>HvGSKs</i> in different plant species.</p></div

Schematic structure of HORVU5Hr1G119790.1 transcript predicted to encode amine oxidase and Glycogen Synthase Kinase (GSK) and HORVU5Hr1G119790.18 transcript annotated as <i>HvGSK4</i>.<i>1</i>.

<p>Enlarged exon10-exon11 fragment with non-transcribed junction between amine oxidase and GSK encoding regions. Indicated are nucleotide coordinates from the current barley genome Hv_IBSC_PGSB_v2.Enlarged exon9-exon12 region of HORVU5Hr1G119790.1 and HORVU5Hr1G119790.18 (<i>HvGSK4</i>.<i>1</i>) transcripts. Indicated are annealing sites of primers and results of amplification using barley gDNA or cDNA as template. Nucleotide alignment of amplicon A and the corresponding fragment of HORVU5Hr1G119790 gene are presented as <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0199364#pone.0199364.s005" target="_blank">S4 Table</a>.</p

Relative expression profile of the 7 <i>HvGSK</i> genes in selected organs and developmental stages of barley plants.

<p>The results represent the mean values and SD of a ratio of the studied gene transcript to the transcript of ADP-ribosylation factor used as an internal reference.</p

Phylogenic tree of <i>GSK</i> genes of <i>Arabidopsis thaliana</i> and <i>Hordeum vulgare</i> used for classification of barley GSK family members.

<p>The phylogeny was reconstructed based on amino acid sequence of kinase domains using maximum likelihood algorithm. GSK protein from <i>Physcomitrella patens</i> (PpGSK) was used as an outgroup.</p

Phenotypes of typical and atypical <i>HvCKX9</i> silent lines.

<p>Typical silent (first left) and variant phenotype (right) of one selected atypical <i>HvCKX9</i> silent line showing long, immature kernels (A) and much bigger maturing grains (B), and not filled mature kernels (C) and only slightly bigger mature grains (D). Typical silent lines are phenotypically not distinguished from control lines.</p

Quantitative analysis of expression patterns of <i>HvCKX</i> genes.

<p>(A) <i>HvCKX1</i>, (B) <i>HvCKX9</i>, (C) <i>HvCKX4</i>, (D) <i>HvCKX5</i>, (E) <i>HvCKX11</i> expression in different tissues and organs of developing plants of two barley cultivars, Golden Promise (left side) and Scarlett (right column). Total RNA was extracted from: 1) 3-day old seedlings, 2) roots from 5-day old seedlings, 3) meristems from 5-day old seedlings, 4) leaves from 5-day old seedlings, 5) developing leaves from 3–4 week old plants, 6) fully developed leaves from 3–4 week old plants, 7) inflorescences 3–4 cm long, 8) inflorescences 6–8 cm long, 9) developing kernels 0 DAP, 10) developing kernels 7 DAP, 11) developing kernels 14 DAP. The level of expression was related to the root  = 1.00 (A) or fully developed leaves  = 1.00 (B,C,D,E).</p

Comparison of means of CKX enzyme activity with the plant height and grain yield in plants silent for <i>HvCKX9</i> T₁.

<p>The bold numbers are significantly different from control values (last row of data) at <i>P</i> <0.05.</p><p>Comparison of means of CKX enzyme activity with the plant height and grain yield in plants silent for <i>HvCKX9</i> T₁.</p

Sequence data of barley <i>HvCKX</i> gene family members published in NCBI databases.

a<p>- annotated by Mameaux et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115729#pone.0115729-Mameaux1" target="_blank">[17]</a> as <i>HvCKX9</i> (99% homology to <i>HvCKX2</i>)</p><p>*- located on chromosome 3H</p><p>** - located on chromosome 2H</p>b<p>- predicted <i>HvCKX</i> gene name</p><p>NI – not identified. Analyzed genes are underlined.</p><p>Sequence data of barley <i>HvCKX</i> gene family members published in NCBI databases.</p

Analysis of inheritance of productivity and other phenotypic traits in subsequent generations.

<p>(A) T₂, T₃ and T₄ lines silent for <i>HvCKX1</i> and (B) T₀, T₁ T₂ and T₃ lines silent for <i>HvCKX9</i>. * - significant differences at <i>P</i><0.05.</p

An example of characteristics of segregating T₁ plants of cv. Golden Promise silent for <i>HvCKX9</i>.

<p>Bars represent relative levels of CKX activity in leaves and 7 DAP spikes, quantitative transcript level in 7 DAP spikes, and silencing coefficient. Grain yield was estimated as a percentage compared to 100% of control GP. The numbers below the columns are transgenic lines: T₀/T₁ plant. * - significant differences at <i>P</i><0.05.</p