Overexpression of Cytokinin Dehydrogenase Genes in Barley (<i>Hordeum vulgare</i> cv. Golden Promise) Fundamentally Affects Morphology and Fertility

<div><p>Barley is one of the most important cereal crops grown worldwide. It has numerous applications, but its utility could potentially be extended by genetically manipulating its hormonal balances. To explore some of this potential we identified gene families of cytokinin dehydrogenases (CKX) and isopentenyl transferases, enzymes that respectively irreversibly degrade and synthesize cytokinin (CK) plant hormones, in the raw sequenced barley genome. We then examined their spatial and temporal expression patterns by immunostaining and qPCR. Two CKX-specific antibodies, anti-HvCKX1 and anti-HvCKX9, predominantly detect proteins in the aleurone layer of maturing grains and leaf vasculature, respectively. In addition, two selected <i>CKX</i> genes were used for stable, <i>Agrobacterium tumefaciens</i>-mediated transformation of the barley cultivar Golden Promise. The results show that constitutive overexpression of <i>CKX</i> causes morphological changes in barley plants and prevents their transition to flowering. In all independent transgenic lines roots proliferated more rapidly and root-to-shoot ratios were higher than in wild-type plants. Only one transgenic line, overexpressing <i>CKX</i> under the control of a promoter from a phosphate transporter gene, which is expressed more strongly in root tissue than in aerial parts, yielded progeny. Analysis of several T1-generation plants indicates that plants tend to compensate for effects of the transgene and restore CK homeostasis later during development. Depleted CK levels during early phases of development are restored by down-regulation of endogenous <i>CKX</i> genes and reinforced <i>de novo</i> biosynthesis of CKs.</p></div

Specific CKX activity in transformed tissues.

<p>Results for two out of seven independent transformants with the <i>Ubi::ZmCKX1</i> construct and two out of five with the <i>PHT::ZmCKX1</i> construct, selected to represent plantlets yielding the most divergent values, obtained from CKX assays with the preferred substrate for ZmCKX1 (iP) and HvCKX9 (iP9G); ND – not determined; CTRL – non-transformed plant regenerated <i>in vitro</i>; WT – plant germinated from wild-type grain.</p

Endogenous CK levels (pmol g⁻¹ fresh weight) in leaves and roots of T1 <i>PHT::ZmCKX1</i> barley plants.

<p>The CKs analyzed included: free bases of <i>trans</i>-zeatin (tZ), <i>cis</i>-zeatin (cZ), dihydrozeatin (DHZ) and <i>N</i>⁶-isopentenyladenine (iP); their N⁹-ribosides (tZR, cZR, DHZR, iPR); <i>O</i>-glucosides (tZOG, cZOG, DHZOG); <i>O</i>-glucoside-<i>N</i>⁹-ribosides (tZROG, cZROG, DHZROG); <i>N</i>⁹-glucosides (tZ9G, cZ9G, DHZ9G, iP9G); and <i>N</i>⁹-riboside-5′-monophosphates (tZR5′MP, cZR5′MP, DHZ5′MP, iPR5′MP); sums of free bases and <i>N</i>⁹-ribosides are considered to comprise the pool of active CKs; mean values with standard deviations obtained from three biological replicates are presented; asterisks indicate significant differences between control and transformed tissue according to Student's unpaired t-tests at P≤0.05; UDL – under detection limit; WT – plants germinated from wild-type grains.</p

Phenotype of T1-generation <i>PHT::ZmCKX1</i> barley transformants.

<p>2-month-old aerial parts of T1-generation plant (<b>A</b>), 4-month-old aerial parts (<b>B</b>) and its root system (<b>C</b>); WT – non-transformed plant. Developing flowers exposed from the flag sheath of the youngest (<b>D</b>) and the oldest tillers (<b>E</b>) of T1 transgenic plants (left, YTT and OTT) and appropriate control plants (right, YTC and OTC). Western blot analysis of two independent 4-month-old T1 PHT2 plants (<b>F</b>); M – marker 60 kDa and 80 kDa, RP – ZmCKX1 recombinant protein <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0079029#pone.0079029-Bilyeu1" target="_blank">[21]</a>, proteins extracted from transgenic (ST) and control (SC) stems and roots (RT, RC).</p

Transcript abundance of the transgene in leaves and roots of T0-generation plants transformed with <i>ZmCKX1</i> (Z2, Z4) or <i>HvCKX9</i> (Ubi 9) genes under control of the <i>Ubi</i> promoter (A).

<p>Transcript abundance of the transgene and <i>PHT1-1</i> gene in leaves and roots of T0-generation plants transformed with <i>ZmCKX1</i> under control of the <i>PHT1-1</i> promoter (<b>B</b>). Transcript abundance of the transgene, <i>PHT1-1</i> gene and the most abundantly expressed endogenous <i>CKX</i> and <i>IPT</i> genes in leaves and roots of T1-generation plants transformed with <i>ZmCKX1</i> under control of the <i>PHT1-1</i> promoter (PHT2 line) (<b>C</b>). Abundance expressed as transcript number per ng of total RNA amplified by qPCR with respect to primer pair efficiency. RNA from two biological replicates was transcribed in two independent reactions, and PCR was performed in duplicate. Mean values with standard deviations are shown.</p

Imunohistochemical localization of barley CKX proteins.

<p>Proteins were detected in paraformaldehyde-fixed tissues using antibodies raised against HvCKX9 (<b>A</b>) and HvCKX1 (<b>C</b>) fragments. Control sections were processed in the same way as samples but omitting incubation with the primary antibody (<b>BD</b>). Mature grains in the hard dough stage (<b>CD</b>) and 10-day-old first leaves (<b>AB</b>) were sectioned on a vibratome. The bar indicates 50 µm.</p