Expression patterns of HvCKX genes indicate their role in growth and reproductive development of barley.

Cytokinin oxidase/dehydrogenase proteins (CKX) are encoded by a multigene family of CKX genes with a varying number of members depending on species. For some of the genes, spectacular effects on grain production in selected cereals have been observed. Despite the fact that partial or full length sequences of most HvCKX genes in barley (Hordeum vulgare) have already been published, in most cases their specific biological functions have not been reported. Detailed expression patterns for five HvCKX genes in different organs/tissues of developing barley plants coupled with analysis of RNAi silent for two genes are presented to test the hypothesis that these expression profiles might indicate their function. Elevated expression for four of them - HvCKX1, HvCKX9, HvCKX4, and HvCKX11 - was found in developing kernels of wild-type plants compared to other tissues. HvCKX5 was mainly expressed in leaf tissue. Lower expression was noted for HvCKX1 in seedling roots and for HvCKX9 in leaves. The documented effect of RNAi silencing of HvCKX1 and a trend for HvCKX9 was higher plant productivity, and the trait was inherited through four generations. Higher plant yield was determined by higher numbers of seeds and spikes. Increased productivity was significantly greater in HvCKX1 silenced plants showing higher relative expression of HvCKX1 in developing kernels of wild-type plants compared to the expression of HvCKX9. Both HvCKX1 silenced T1 seedlings of cv. Golden Promise and the newly transformed breeding line STH7308 showed greater root mass, but this trait was not inherited in the next generation. Similarly HvCKX9 silenced T1 seedlings exhibited greater plant height without inheritance in the next generation. It is suggested that these effects were not inherited because of compensation by other genes co-ordinately regulating reproductive development. One line with untypically changed, inherited phenotype, which was selected from several dozen silenced lines showing stable and common phenotypes is presented

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

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

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

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