Plant Physiology Preview. Published on June 5, 2018, as DOI: /pp

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1 Plant Physiology Preview. Published on June 5, 2018, as DOI: /pp Short title: Root Enhancement in Barley Corresponding author Prof. Dr. Thomas Schmülling Institute of Biology/Applied Genetics Dahlem Centre of Plant Sciences (DCPS) Freie Universität Berlin Albrecht-Thaer-Weg 6 D Berlin, Germany tschmue@zedat.fu-berlin.de Phone: Fax: Root Engineering in Barley: Increasing Cytokinin Degradation Produces a Larger Root System, Mineral Enrichment in the Shoot and Improved Drought Tolerance Eswarayya Ramireddy 1,2, Seyed A. Hosseini 3,5, Kai Eggert 3, Sabine Gillandt 1, Heike Gnad 4, Nicolaus von Wirén 3 and Thomas Schmülling 1 1 Institute of Biology/Applied Genetics, Dahlem Centre of Plant Sciences (DCPS), Freie Universität Berlin, Albrecht-Thaer-Weg 6, D Berlin, Germany 2 Indian Institute of Science Education and Research Tirupati, Biology Division, Tirupati , Andhra Pradesh, India 3 Molecular Plant Nutrition, Leibniz-Institute of Plant Genetics and Crop Plant Research, Corrensstr. 3, D Stadt Seeland OT Gatersleben, Germany 4 Saaten-Union Biotec GmbH, Am Schwabeplan 6, D StadtSeeland OT Gatersleben, Germany One sentence summary: Root-specific expression of a cytokinin-degrading CKX gene in barley roots causes formation of a larger root system leading to higher element content in shoot organs and improved drought tolerance. 1 Copyright 2018 by the American Society of Plant Biologists

2 Footnotes: E.R. and T.S. designed experiments and coordinated the project; E.R. and S.G. carried out molecular and phenotypic analysis of barley plants; H.G. produced transgenic barley plants; S.A.H carried out seed element and metabolite analyses, K.E. carried out cytokinin measurements, N.v.W. designed experiments and analyzed data, E.R. and T.S. wrote the article with contributions of all authors. Funding: Work has been financed by grants of the German Federal Ministry for Education and Research (BMBF) to T.S. and H.G. in the frame of the PLANT-KBBE program and the project ROOT: Root enhancement for crop improvement. Corresponding author 5 Present address: Plant Nutrition Department, Centre Mondial de I lnnovation Roullier, 18 Avenue Franklin Roosevelt, F Saint Malo, France 2

3 ABSTRACT Root size and architecture are important crop plant traits, as they determine access to water and soil nutrients. The plant hormone cytokinin is a negative regulator of root growth and branching. Here, we generated transgenic barley (Hordeum vulgare L.) plants with an enlarged root system by enhancing cytokinin degradation in roots to explore the potential of cytokinin modulations in improving root functions. This was achieved through root-specific expression of a CYTOKININ OXIDASE/DEHYDROGENASE (CKX) gene. Enhanced biomass allocation to roots did not penalize shoot growth or seed yield, indicating that these plants were not source-limited. In leaves of transgenic lines, the concentration of several macro- and micro-elements was increased, particularly those with low soil mobility [phosphorus (P), manganese (Mn), zinc (Zn)]. Importantly, seeds contained up to 44% more Zn, which is beneficial for human nutrition. Transgenic lines also demonstrated dampened stress responses to long-term drought conditions, indicating lower drought sensitivity. Taken together, this work demonstrates that root engineering of cereals is a promising strategy to improve nutrient efficiency, bio-fortification and drought tolerance. Keywords: barley, bio-fortification, CKX, cytokinin, cytokinin oxidase/dehydrogenase, drought resistance, Hordeum vulgare L., mineral nutrition, plant development, root system architecture 3

4 INTRODUCTION The size and architecture of root systems are important traits of crop plants. Plant roots perform many essential functions including taking up water and nutrients, storing reserves, anchoring the plant to the soil and establishing biotic interactions in the rhizosphere. The root system s size and architecture determine its ability to fulfil these diverse functions. This is relevant for crop plants, as shortages of water and nutrients limit growth and grain yield in many agro-ecosystems (Lynch, 1995). Drought accounts for more loss in plant productivity than any other abiotic factor, and climate change models predict an increase of drought stress in several crop production regions worldwide (Hochholdinger, 2016). Another critical yield-limiting factor is the access of roots to soil nutrients. Increasing crop yields and expansion of agriculture to marginal lands increases demand for soil nutrients, which needs to be covered by fertilization. Fertilizers are expensive and especially nitrate and phosphate leaching or runoff cause eutrophication of surface waters and contamination of groundwater. To address these problems, plant breeding aims to develop varieties with improved nutrient use efficiency and improved drought tolerance. One way to improve nutrient and water use efficiency and to secure plant productivity under non-optimal conditions may be achieved by improving the root system of crop plants (Lynch and Brown, 2012; Comas et al., 2013; White et al., 2013; Rogers and Benfey, 2014; Koevoets et al., 2016). In general, root length and root density are positively correlated with mineral element uptake, particularly for elements with limited solubility (Marschner, 2002). Root architecture also determines access to water, and under certain conditions a correlation has been found between root system size and tolerance to drought stress (Price et al., 2002; Tuberosa et al., 2002; Comas et al., 2013). For example, drought-tolerant rice varieties have been found to form a deeper and more highly branched root system than drought-sensitive varieties (Price and Tomos, 1997). Other studies have revealed a positive correlation between root traits and crop performance or grain yield under drought (de Dorlodot et al., 2007; Kell, 2011; Uga et al., 2013; Hufnagel et al., 2014; Meister et al., 2014). Consequently, root architectural traits optimizing soil exploration in time and space are among those traits that are considered to be relevant in crop breeding programs (Lynch and Brown, 2012; Comas et al., 2013; White et al., 2013; Rogers and Benfey, 2014; Koevoets et al., 2016). Optimizing the root system by classical breeding strategies requires detailed measurements of roots, which are hidden below ground. In addition, root system architecture is a complex trait, governed by many intrinsic and extrinsic factors and involving numerous genes (Lynch and Brown, 2012). These difficulties make it challenging to breed specifically for improved root systems. However, a targeted approach to improve root systems is desirable, not only for breeding purposes but also to study the functional relevance of the size of the root system. Transgenic lines and their non-transgenic counterparts would be 4

5 near-isogenic lines differing in only one or two genes, which is advantageous for functional studies. The plant hormone cytokinin (CK) has been recognized as a major negative regulator of root development. In Arabidopsis (Arabidopsis thaliana), CK regulates the elongation of primary roots (Werner et al., 2003), the initiation of lateral roots (Laplaze et al., 2007; Chang et al. 2013) and acts as a positional cue regulating the distance between lateral roots (Bielach et al., 2012; Chang et al., 2015). Different approaches have been used to reduce the CK status of Arabidopsis, including overexpression of CK-degrading CK oxidase/dehydrogenase (CKX) genes (Werner et al. 2001, Werner et al., 2003), mutation of CK synthesis genes (Miyawaki et al., 2006), mutation of CK receptor genes (Riefler et al., 2006) or suppression of the CK signaling pathway (Mason et al., 2005; Heyl et al., 2008). Collectively, these studies have shown that under standard growth conditions the root CK status is above the optimal level for root growth. Furthermore, proof has been obtained in dicotyledonous model plants that increasing CK degradation in roots by root-specific expression of a CKX gene leads to formation of an enhanced root system, causes increased accumulation of several micro- and macronutrients in the aerial plant parts and improves survival under harsh drought conditions (Werner et al., 2010). This approach demonstrated that a single dominant gene can be used to regulate a complex trait. CK is also involved in regulating plant responses to the deficiency of several essential nutrients, including those of phosphorus (Franco-Zorilla et al., 2002, 2005), sulphur (Maruyama-Nakashita et al., 2004), iron (Séguéla et al., 2008), sodium (Mason et al., 2010), potassium (Nam et al., 2012) or to the toxicity of arsenic (Mohan et al., 2016). Since the expression of CK-related genes is responsive to soil conditions, indicating that the environment influences cellular CK homeostasis (Ramireddy et al., 2014), an altered CK status of the root may improve the acquisition of soil nutrients (Werner et al., 2010). Much less is known about CK and its potential usefulness to manipulate the root system in cereal plants, which would be a primary target for application because of their agricultural relevance. Cereal plants have more complex root systems than Arabidopsis, as these consist of embryonic primary and seminal roots and post-embryonic crown roots (Rogers and Benfey, 2015). Monocot plants such as maize (Zea mays L.) and rice (Oryza sativa L.) possess a similar set of CK genes as dicot plants (Chu et al., 2011; Tsai et al., 2012), and some of these genes have been shown to be involved in regulating crown root formation in rice (Kitomi et al., 2011; Gao et al., 2014; Zhao et al., 2015). The inhibition of lateral root initiation but stimulation of lateral root elongation by CK in rice (Rani Debi et al., 2005) suggests that CK functions are generally quite similar between monocots and dicots, but that there might also be key differences that must be identified to inform efforts to improve root traits in grasses. Also the higher content and activity of the generally less active 5

6 cis-zeatin (cz)-type CKs as compared to trans-zeatin (tz)-type CKs in some monocot plants suggests that dicot and monocot plants differ in some aspects of CK metabolism, transport or signaling (Lomin et al., 2011; Kudo et al., 2012). We selected barley (Hordeum vulgare L.), which is an important European cereal crop species, for our root-engineering approach. The barley root consists of several seminal roots derived from the embryo and a nodal root system derived from the nodes at the base of the main stem or of the tillers. Previous attempts to manipulate the barley root system by ectopic expression of CKX genes have proven difficult as strong detrimental effects on shoot development and reproduction could not be avoided (Mrizová et al., 2013; Pospíšilová et al., 2016). This indicated that a successful approach may depend on restricting the manipulation of CKX gene expression to roots. We, therefore, attempted here to enhance the root system of barley plants by ectopic expression in roots of CKX genes previously characterized in Arabidopsis. As it was a priori not obvious which CKX enzyme from Arabidopsis would be most suitable to achieve root enhancement in barley, we compared the two prototypic enzymes CKX1 and CKX2, which in Arabidopsis affect different CK pools because of their distinct subcellular localization and substrate specificities (Werner et al., 2003; Galuszka et al., 2007). CKX1 and CKX2 belong to two different evolutionary branches of the CKX protein family that had formed before monocots and dicots diverged (Schmülling et al., 2003). In the present work, we show that it is possible by genetic engineering of CK breakdown to generate barley plants that have an enhanced root system but largely unaffected shoot development. Comparison to their untransformed counterparts revealed the increased accumulation of several soil nutrients in their leaves and seeds as well as a reduced sensitivity to long-term drought conditions. RESULTS Identification and Validation of Root-Specific Promoters for Root Engineering in Barley For altering root growth and development by enhanced expression of CKX genes, the use of a root-specific promoter appeared indispensable (Werner et al., 2010). For our experiments in barley, we searched for a root-specific promoter in rice, because of the close genetic relationship between rice and barley and the availability of transcriptome data and genome sequences from rice. By mining publicly available transcriptome data (Winter et al., 2007; Hruz et al., 2008; Sato et al., 2011), we identified several rice genes that were exclusively or preferentially expressed in root tissues at different developmental stages, including RETROTRANSPOSON PROTEIN (RET; LOC_Os10g31730), EXPRESSED PROTEIN (EPP; LOC_Os04g11040) and PEROXIDASE PROTEIN (PER; LOC_Os03g25330) 6

7 (Supplemental Fig. S1A). All three genes were expressed in roots under different growth conditions and at different developmental stages, including vegetative and reproductive growth stages of field-grown rice cultivars (Sato et al., 2011). In order to validate root-specific expression of these genes, reverse transcription quantitative PCR (RT-qPCR) analysis was carried out with RNA from different tissues of soil-grown rice. In roots, transcript levels of RET, EPP or PER were > 2000-, 300-, or 10-fold higher, respectively, than in any of the other organs tested (Supplemental Fig. S1B). Subsequently, expression of the GUS reporter gene under the control of ~2 kb of the EPP and PER promoters was tested in Arabidopsis. GUS staining was found almost exclusively in roots at different developmental stages of Arabidopsis (Supplemental Fig. S2). Within the primary root, expression was restricted to the transition zone and the vascular tissue, and no expression was observed in primary and lateral root meristems. These results indicated that the EPP and PER promoters mediate root-specific expression in monocotyledonous and dicotyledonous species, thus being suitable to drive CKX gene expression in our approach with transgenic barley. Generation of Transgenic Barley Plants with Increased CKX Activity in Roots For root engineering in barley, we used two prototypic CKX genes of Arabidopsis, CKX1 and CKX2. The two CKX gene products, CKX1 and CKX2, differ in their subcellular localization and biochemical characteristics. A tagged CKX1 protein was shown to predominantly localize to the endoplasmic reticulum (ER) (Niemann et al., 2018) while a CKX2-GFP fusion protein localized to the ER and possibly also to the apoplastic space (Werner et al., 2003; Zürcher et al., 2016). Furthermore, the CKX1 protein preferred CK ribosides and N 9 -glucosides as substrates, while N 6 -isopentenyladenine (ip) and ip riboside (ipr) were preferred by CKX2 (Galuszka et al., 2007). Root-specific expression of either CKX gene strongly enhanced root growth in tobacco (Nicotiana tabacum) and Arabidopsis (Werner et al., 2010) but only enhanced CKX1 expression had a strong negative impact on shoot growth (Werner et al., 2001, 2003). Transgenic barley plants expressing CKX1 or CKX2 under control of the three rootspecific promoters pepp, pper and pret were generated using Agrobacterium-mediated transformation of the spring barley variety Golden Promise (Bartlett et al., 2008). Expression of the transgenes was tested by RT-qPCR in several independent homozygous lines of the T3 generation. Since pepp:ckx lines were mostly used for further analyses we document the characterization of these lines in more detail. Root specificity of CKX2 transgene expression in the line pepp:ckx2-72 was confirmed 5, 14 and 21 days after germination (DAG), whereas in the shoot tissue no expression was detected (Fig. 1A). For further studies, two independent transgenic barley lines with high expression levels were selected for each of the two CKX homologs, i.e. pepp:ckx1-4, pepp:ckx1-109, pepp:ckx2-16 and 7

8 pepp:ckx2-72. In all four lines, transcript levels of the transgene were high in the roots (Fig. 1B). Subsequently, CKX activity was tested in three-week-old soil-grown transgenic plants expressing the CKX2 gene and compared to the wild type. The analysis was limited to CKX2, as CKX1 activity is very low in barley and difficult to quantify (see also Galuszka et al., 2007). Both pepp:ckx2 lines showed >7.5-fold higher CKX activities in roots when compared to roots of untransformed plants (Fig. 1C). An increase in CKX activity was also observed in the shoot tissue of transgenic lines, but there the enzyme activity was only about 6-11% of the activity found in roots (Fig. 1C). Quantification of CK metabolites in the roots of three-week-old plants of the transgenic barley lines revealed a trend of lower levels of CK metabolites compared to wild type (Fig. 1D; Supplemental Table S1). In particular, the root concentration of the free base ip, which was the only bioactive CK detected, was only between 21% and 47% of the concentration in wild-type roots. The total content of ip-type and cz-type CKs in transgenic roots was in the range of 57-89% of that of the wild type. The reduction was stronger for tz-type CKs: three transgenic lines contained 9-38% of the concentration of the wild type, while the line pepp:ckx1-4 contained 87% of tz-type CKs in its roots (Fig. 1D; Supplemental Table S1). A large part of the reduction was due to a lower concentration of CK sugar conjugates, which are biologically inactive CK storage forms (Sakakibara, 2006) and represented the bulk of CK metabolites (Supplemental Table S1). The strong reduction of their concentration indicated reduced availability of the physiologically active form tz, which by itself was below the detection level. The total concentration of cz-type CKs was slightly lower in roots of the transgenic lines. In shoots, the total concentration of ip-type CKs was unchanged, while the concentration of tz-type CKs in lines 16 and 72 was about two-fold higher than in the wild type but unchanged in two other lines; in this case, this difference was also mainly due to an altered concentration of the corresponding conjugates (Supplemental Table S1). Transgenic Barley Plants Exhibit Enlarged Root Systems We then investigated whether enhanced CKX expression in roots of transgenic barley would alter plant growth. Visual inspection of roots from two-week-old hydroponically-grown plants indicated that the size of the root system was apparently increased in transgenic plants compared to the wild type (Fig. 2A). Quantitative analysis revealed an increase of the total root length by 24-70% and of the total root surface area by 12-50% in transgenic plants compared to wild type (Fig. 2B and 2C). Root biomass of transgenic plants was increased by up to 47% in comparison to wild-type roots (Fig. 2D). In contrast, the shoot biomass of the transgenic lines was comparable to that of the wild type, except for line pepp:ckx1-109, which showed a 15% increase in shoot biomass (Fig. 2E). The differential root growth increased the root-to-shoot biomass ratio by 16-50% in the transgenic lines. The analysis of 8

9 three-week-old plants grown in hydroponics confirmed that transgenic plants developed a larger root system size with increased biomass but essentially without reduction in shoot biomass (Supplemental Fig. S3A-C). Growth of two transgenic lines was also tested in soilfilled rhizoboxes (Supplemental Fig. S3D). Also under these conditions, root size and root-toshoot biomass ratio of these plants was increased by about 30% and 35-75%, respectively, as compared to the wild type (Supplemental Fig. S3E-G). Notably, pret:ckx and pper:ckx transgenic lines also developed a larger root system (Supplemental Fig. S4). Root-Specific Expression of CKX Does Not Cause Yield Penalty Next, we analyzed whether the increased root size of transgenic plants would have an impact on seed yield. Visual inspection of three-month-old soil-grown transgenic plants confirmed their enhanced root system at this late developmental stage (Fig. 3A). However, growth and development of the shoots of these plants appeared to be similar to wild-type plants. For example, plant height and the number of days to reach the heading stage were similar in all lines (data not shown). Likewise, shoot biomass (on a dry-weight basis) of 70- day-old transgenic plants was comparable to that of the wild type (Fig. 3B). Furthermore, yield-related traits like the number of tillers per plant (Fig. 3C), the number of ears per plant (Fig. 3D), total grain yield per plant (Fig. 3E) and the hundred-kernel weight (Fig. 3F) were not significantly different from the wild type at p < Only line pepp:ckx1-4 showed a significantly (p < 0.01) larger number of ears per plant, however, this increase did not affect total grain yield (Fig. 3E). Taken together, root-specific expression of CKX genes caused root enhancement but did not significantly affect shoot growth or seed yield in the transgenic lines. Root-specific Expression of CKX Enhances Mineral Element Accumulation in Leaves and Seeds One important function of roots is to acquire mineral elements from the soil. The ability of roots to efficiently explore and exploit the available soil volume is particularly relevant for nutrients with low soil mobility. Therefore, enhanced growth of roots might contribute to improved extraction of such nutrients from the soil. To study whether the increased root size had an impact on element content of aboveground plant parts, we determined the concentrations of 16 different elements in leaves and seeds of the transgenic lines. In leaves from eight-week-old soil-grown transgenic plants, concentrations of numerous mineral elements were higher than in wild-type plants, and there was a stronger increase in lines expressing CKX2 than in those expressing CKX1 (Fig. 4A and Supplemental Table S2). Of the five macro-elements [phosphorus (P), potassium (K), sulfur (S), magnesium (Mg), calcium (Ca)] the concentrations of P and S were increased by 13-53% and 17-45% in three 9

10 of the four lines, respectively (Fig. 4A and 4B). In contrast, leaf concentrations of K tended to be slightly reduced, i.e. by 5-13% in all four transgenic lines (Fig. 4 and Supplemental Table S2). Similarly, the concentrations of several microelements were increased in at least three of the four transgenic lines, including copper (Cu, 20-28%), manganese (Mn, 51-70%) and zinc (Zn, 7-54%). The concentrations of other elements [calcium (Ca), iron (Fe), boron (B)] were increased compared to wild type as well, but the difference was usually only statistically significant either in two lines, pepp:ckx1-4 and pepp:ckx2-72 or in pepp:ckx2-72 alone (Fig. 4 and Supplemental Table 2). The enhanced ability of transgenic lines to accumulate nutrients was also evaluated in plants grown under drought stress. Under drought conditions, nutrient uptake is generally lower due to a decrease in transpiration rate and reduced nutrient uptake into root cells (Levitt, 1980). In our experiment, drought stress did not strongly affect the concentrations of mineral elements in any of the genotypes (Supplemental Table S3). Transgenic plants grown under well-watered or drought stress conditions showed enhanced accumulation of several elements to a similar extent as in the experiment described above although they were grown in a different soil. In particular, enhanced accumulation of Mn ( %) and P (+18-35%) in both transgenic lines under all conditions indicated that this is a stable trait. We then investigated whether the increase in nutrient accumulation found in leaves is reflected by a similar increase in seeds. To this end, we analyzed element concentrations in dried seeds of the two CKX2-expressing lines, as these showed the highest leaf element accumulation, and the wild type. In contrast to leaves, the concentrations of most of the elements were similar in all lines (Supplemental Table S4). However, the concentrations of Ca, Cu and Zn were consistently increased in seeds of transgenic plants (Fig. 5A and Supplemental Table 4). The increase was significant for Zn in both lines (26% and 44%, respectively), whereas the increases for Ca and Cu were significant only in line pepp:ckx2-72 (Fig. 5B; Supplemental Table S4). Taken together, in seeds of transgenic barley plants an increased concentration was found for a subgroup of elements that were increased in leaves. Transgenic Plants Withstand Long-Term Drought Better than Wild Type As crop plants frequently experience water stress, we explored the response of transgenic barley plants to long-term drought conditions. The two pepp:ckx2 transgenic lines which showed the strongest root growth were compared to wild type. Drought stress was imposed for two weeks on plants having eight to nine tillers by maintaining moisture of the growth substrate at 10% using a soil moisture meter, corresponding to 20-25% of field capacity. Several parameters indicating stress responses were monitored, including stomatal conductance, transpiration rate and carbon assimilation, all impacting photosynthesis. 10

11 Under well-watered (control) conditions, stomatal conductance and transpiration rate were not significantly different between transgenic and wild-type plants (Fig. 6A and 6B). Relative to control conditions, water deficit reduced stomatal conductance and transpiration rate in wild-type plants down to 11% and 18%, respectively. In transgenic plants, stomatal conductance was reduced to 25-29% and transpiration rate was reduced to 30-32% of control conditions (Fig. 6A and 6B). Similarly, the CO 2 assimilation rate was reduced to 13% of the control conditions in wild type but only to 36-45% in the transgenic lines (Fig. 6C). Wild-type plants and transgenic plants contained a similar concentration of sugars under control conditions and all genotypes showed an increase under drought, which was significantly stronger for wild type (Fig. 6D-F). Accumulation of sugars is important for osmotic adjustment under drought stress (Seki et al., 2007; Todaka et al., 2017). Together these results indicated that transgenic plants withstood prolonged water deficit better than wild-type plants, which is particularly evident from the higher CO 2 assimilation rate in the transgenic plants. Next we measured the concentrations of abscisic acid (ABA), which is a master regulator of the drought stress response (Shinozaki and Yamaguchi-Shinozaki, 2007), and its catabolic products phasic acid (PA) and dihydrophasic acid (DPA). PA and DPA tend to accumulate and can therefore be seen as an indicator for the length of a stress period. Under control conditions, the steady state levels of ABA and its catabolites were low and similar or slightly lower in transgenic as compared to wild-type plants (Fig. 7A). Drought caused an 11-fold increase in the ABA level of the wild type and a four- to five-fold increase in transgenic plants (Fig. 7A). The accumulation of PA and DPA in response to drought was lower in the transgenic lines than in the wild type (Fig. 7B and 7C). Gene expression analysis showed that transcript levels of the key gene in ABA synthesis, 9-cis-EPOXYCAROTENOID DIOXYGENASE2 (HvNCED2), were much lower in transgenic plants than in the wild type (Fig. 7E). Under drought conditions, the HvNCED2 transcript level in the two transgenic lines remained about 20-fold lower than in wild-type plants (Fig. 7E). The transcript levels of the gene ABA-8 -HYDROXYLASE (HvABA-8 -OH), coding for a protein that mediates the key step in ABA degradation, were also lower in the transgenic lines but the differences were not as large as for HvNCED2 (Fig. 7F). These results showed that CKX expression in roots led to a reduced accumulation of ABA, PA and DPA under drought, which was reflected by dampened ABA metabolism as indicated by the transcript levels of key genes. It has been shown that several plant species accumulate certain amino acids upon exposure to abiotic stress, which might thus serve as metabolic stress markers (reviewed in Krasensky and Jonak, 2012). A comparison of free amino acid concentrations in the shoots of plants grown under control or drought conditions revealed that except for methionine (Met), the concentrations of all measured amino acids were several-fold higher in transgenic 11

12 compared to wild-type plants (Table 1). Under control conditions, the total concentration of proteinogenic amino acids was about 3-fold higher in transgenic plants in comparison to the wild type. In contrast, the concentration of methionine was reduced by about 8-30-fold in transgenic plants. Notably, the steady state level of the non-proteinogenic amino acid γ- aminobutyric acid (GABA) was also 4-5-fold higher in transgenic plants. Drought treatment caused a 6.6-fold increase in total amino acid content in the wild type, whereas it was only ~1.7-fold higher in the transgenic plants (Table 1). The three amino acids asparagine (Asn), serine (Ser), and threonine (Thr) were of particular interest as their concentrations have been shown to correlate positively with the performance of rice under drought (Degenkolbe et al. 2013). Under control conditions, their concentrations were significantly higher in transgenic plants than in wild-type plants. Following drought stress, their concentrations increased by 30-fold (Asn), 14-fold (Ser) and 6.8-fold (Thr), respectively, in the wild type but only ~2.5-, 1.5- and ~2-fold, respectively, in the transgenic lines (Table 1). Proline (Pro) is known to accumulate in considerable amounts in response to abiotic stress like drought, salt or osmotic stress (Sharma et al., 2011; Kavi Kishor and Sreenivasulu, 2014). Under control conditions, the steady state level of Pro was 5-6-fold higher in transgenic than in wild-type plants (Fig. 7D). However, Pro accumulated massively (~620-fold increase) in the wild type in response to drought stress. In transgenic plants, Pro accumulation increased only by 20- to 50-fold compared to the control conditions and thereby remained 40-80% lower than in the wild type (Fig. 7D). Pro homeostasis depends on HvP5CS1 (Δ1-pyrroline-5-carboxylate synthetase1), which is involved in the biosynthesis of Pro from glutamate (Verslues and Sharma, 2010). Examining its transcript levels showed that following drought stress, HvP5CS1 mrna levels increased strongly in all lines, but remained at lower levels in the transgenic lines under both control and drought stress conditions (Fig. 7G). DISCUSSION CKX Transgene Expression is a Valuable Approach for Genetic Engineering of the Root System The present work showed that it is possible to generate barley plants with an enhanced root system by ectopic expression of a single CKX gene without affecting shoot development. Apparently, the enlarged root system was formed without metabolic costs for the shoot, as plant height, heading time, shoot biomass, number of tillers and ears per plant, hundredkernel weight and total grain yield were similar in the transgenic lines and control lines. This indicates that these plants were not source-limited and the carbon fixed by the shoot was sufficient to support stronger root growth. This is an important result strengthening the prospects of using root enhancement as a strategy to improve crop plant performance (de 12

13 Dorlodot et al., 2007; Meister et al., 2014; Rogers and Benfey, 2015; Hochholdinger, 2016; Koevoets et al., 2016). It is in contrast to the long-held view that carbon fixation becomes a limiting factor when plants invest more carbon into root biomass. In such a case, enhanced root growth would establish a competing sink that supports root growth at the expense of shoot development. The present results support the idea that the capacity for tissue formation and growth (sink strength) may be the limiting factor for growth rather than the provision of carbohydrates produced by photosynthesis. Therefore, the data are consistent with the suggested shift of paradigm from a source- to a sink-oriented view when looking at endogenous determinants of plant growth (Körner, 2015). Following this view, the CK concentration in roots of wild-type plants may be considered as higher than optimal for strong root growth. Lowering the concentration of CKs by genetic engineering suppresses the negative impact of CKs on root development and thus enhances sink strength of the root. In Arabidopsis the combination of root-specific CKX gene expression with the expression of transgenes enhancing shoot growth led to an increase of overall plant growth (Vercruyssen et al., 2011). This suggests that transgenic approaches may be used in a combinatorial manner for the growth promotion of below- and aboveground organs. To genetically engineer plants with a root-specific enhancement of CK breakdown the careful selection of a suitable promoter to regulate CKX gene expression is of great importance. We compared three root-specific promoters of rice to drive CKX gene expression and found the EPP promoter to be the most suitable one. However, the pper:ckx and pret:ckx fusion constructs yielded transgenic plants with an enhanced root system and without provoking detrimental effects on shoot growth. Previous attempts to direct CKX gene expression to roots of monocot plants led to plants that formed smaller shoots with severely compromised fertility (Pospíšilová et al., 2016) or plants that were infertile (Mrízová et al. 2013), presumably because the chosen promoters lacked specificity. Root-specific expression of OsCKX4 in rice also caused the formation of a stronger root system but shoot traits were not documented (Gao et al., 2014). Ectopic expression of both prototypic CKX enzymes of Arabidopsis yielded qualitatively similar effects, but CKX2 had a stronger impact on root system size and element accumulation than CKX1. For example, total root length of the plants expressing CKX2 was increased by 60-70%, whereas it was only 20-40% in the plants expressing CKX1. Such a difference between the two genes has also been noted in Arabidopsis and tobacco (Werner et al., 2010), suggesting that the affected subcellular CK pools have a distinct regulatory and apparently evolutionary conserved impact on these traits. This indicates that despite different subcellular distribution and CK metabolite profiles in Arabidopsis and barley (Jiskrová et al., 2016) there must be a common impact of CKX enzymes on the composition or pool sizes of bioactive CKs. 13

14 Root-Specific Expression of CKX Causes Enhanced Element Accumulation in Leaves and Seeds The enhanced root growth in transgenic lines contributed to an improved uptake of several elements from the soil and their increased accumulation in shoot organs. In leaves of transgenic barley plants, the concentrations of several essential elements, in particular of P, Mn and Zn, were significantly increased by up to > 100% in at least three of the four analyzed transgenic lines. Enhanced accumulation of these elements was confirmed in two different substrates and under different growth conditions (well-watered vs. drought stress), indicating that this was a stable trait. Among the two different CKX genes, CKX2 expression had a stronger impact on the accumulation of additional elements including sulfur. Increased uptake of these elements is beneficial as they are limiting in numerous agricultural soils and their availability may limit low-input agriculture. Therefore the development of crop genotypes with root traits increasing element acquisition should increase growth and yield on infertile soils (White et al., 2013). For example, it has been shown for transgenic tobacco plants with an enhanced root system that their accumulation of higher levels of Mg correlates with increased chlorophyll content and better growth on media with low Mg content (Werner et al., 2010). The increased element accumulation in transgenic barley is consistent with the view that among the root traits, total root length is key in the acquisition of sparingly soluble elements (White and Broadley, 2009; White and Greenwood, 2013). Indeed, it has been shown in rice that lateral roots, which contribute the largest portion to total root length, contribute significantly to the acquisition of P, Mn and Zn (Liu et al., 2013). However, not all elements respond in the same way to an enhanced root system. In fact, for most of them the accumulation in leaves was not altered, suggesting that besides a larger soil volume exploited by the enhanced root system of CKX-overexpressing plants additional factors must play a role. The involvement of CK in regulating the response to the availability of P, S, Fe, Na and K is well known (Franco-Zorilla et al., 2002, 2005; Maruyama-Nakashita et al., 2004) suggesting that besides better exploitation of the soil space by the larger root system an altered response to P and S availability may have contributed to their increased uptake. Increased expression of genes encoding transporters for phosphate, sulfate, Mn and Zn, as was found for CK-deficient roots of Arabidopsis (Werner et al., 2010; Brenner and Schmülling, 2012), could make a relevant contribution. The recent finding that CK regulates the formation of passage cells in the Casparian strip, which has an impact on the transfer of soil-derived elements to the vasculature (Andersen et al., 2018), suggests an additional possibility in which an altered root CK status may influence the nutrient uptake. Altered root exudation of element-mobilizing compounds or altered interaction of CK-deficient roots with 14

15 root microbiota (Cosme et al., 2016) may also play a role. However, to what extent these different factors might contribute to enhanced element acquisition is unclear at present. Notably, root-specific CKX gene expression in the dicots Arabidopsis and tobacco caused the accumulation of the same type of elements in leaves as in barley, which suggests that their acquisition depends on common mechanisms which appear to be evolutionarily stable. The reasons why such mechanisms linked to the hormone CK are under selection remain unknown. An Increase in Seed Zn Content Contributes to Biofortification A specific and important aspect of Zn accumulation is that this element not only strongly and consistently increased in leaves but also in seeds, where it can make a valuable contribution to human nutrition. This is relevant as insufficient uptake of Zn by humans causes malnutrition and related health disorders in more than two billion people worldwide (WHO, 2016). Zn deficiency is particularly widespread in developing countries, where nutrition depends mainly on plant-based diets and where lack of Zn contributes significantly to infant mortality (White and Broadley, 2011). Therefore, several breeding programs have been initiated to increase the Zn content in cereal grains (Palmgren et al., 2008; White and Broadley, 2011; Borrill et al., 2014). Our results showed that root enhancement induced by CK deficiency increased the accumulation of Zn in seeds by 26-44%. This increase raised the concentration of Zn from approximately 31 mg kg -1 in wild type to mg kg -1 in seeds of both tested transgenic lines. This is even more than the target Zn concentration of 38 mg kg -1, as set by the HarvestPlus program for wheat (Triticum aestivum) grains (White and Broadley, 2011). For comparison, the enrichment of Zn in maize, rice or wheat grains that was achieved by application of Zn-enriched fertilizers to the soil was in the range of 23%, 7% or 19%, and by foliar application in the range of 30%, 25% or 63%, respectively (Joy et al., 2015). Lonergan et al. (2009) identified in barley several quantitative trait loci (QTLs) that increased the grain Zn content by 53-75%. Considering the costs required for Zn fertilization, achieving Zn enrichment through root enhancement as described here presents a cost-effective and sustainable strategy contributing to genetic biofortification, which could be used in a complementary manner with other efforts (Borrill et al., 2014). In another transgenic approach, endosperm-specific expression of the Zn transporter gene, METAL TOLERANCE PROTEIN1 HvMTP1 doubled Zn accumulation in the endosperm of barley plants, which was most likely conferred by enhanced vacuolar Zn loading (Menguer et al., 2017). Future studies should reveal the molecular mechanism(s) underlying Zn biofortification in CKX transgenic plants and also study tissue localization and chemical speciation of Zn, which affect Zn bioavailability for human consumption. 15

16 Transgenic Plants with Enhanced Root System Size are Less Sensitive to Drought Stress Several parameters indicate that the transgenic barley lines generated here were less sensitive to drought stress. The first response to drought is a reduction in stomatal conductance to decrease water loss by transpiration (Medrano et al., 2002). However, this response impedes plant growth as it restricts CO 2 entry and consequently lowers the photosynthetic rate. For optimal growth under drought, a compromise between carbon assimilation and water transpiration has to be reached. In the present study, transgenic barley plants displayed a fold higher stomatal conductance and, accordingly, an almost 2-fold higher carbon assimilation rate compared to wild type plants under long-term drought conditions. Apparently, such an effective drought avoidance mechanism may at least be partially attributed to the enhanced root system. Several root traits, including the biomass, length, density and depths of roots, have been correlated previously with drought tolerance in different crop species and are proposed to contribute to plant performance under drought conditions (Meister et al., 2014; Kashiwagi et al., 2015; Gagné-Bourque et al., 2016). Model species with larger root system are more tolerant to drought as well (Werner et al., 2010; Macková et al., 2013), which may be partially caused by the role of CK in regulating the response to drought (Nguyen et al., 2016). Interestingly, a recent report shows that root growth is required to sense and inform about the availability of water and induce root branching in the search for water (Robind and Dinneny, 2018). It could be that continued growth of roots of CKX-modulated lines facilitates this process and improves access to water. Noteworthy, CK can also act directly in leaves to affect stomatal behavior. Thus, increasing CK production in leaves during periods of severe drought stress caused drought tolerance (Rivero et al., 2007). Drought induces the accumulation of ABA, as this hormone has an important function in orchestrating the response to drought stress (Verslues, 2016). An increase in ABA can be regarded as an indicator of drought stress. In both the CKX-overexpressing lines and the wild type, the concentrations of ABA and its catabolites PA and DPA increased under experimental drought conditions. However, the concentrations reached in the transgenic lines were only about half of those in the wild type, indicating that the former experienced a weaker stress level. Such a lower ABA accumulation might at least be partially due to a lower basal expression level as well as a reduced response to drought of the ABA biosynthesis gene HvNCED2 and the ABA catabolism gene HvABA-8 -OH (Fig. 7). It has been shown in different perennial grass species that a lower level of ABA accumulation correlates with drought tolerance (Wang et al., 2003; DaCosta and Huang, 2007). This could be due to the avoidance of detrimental effects of increased ABA levels on photosynthesis and growth 16

17 (Sreenivasulu et al., 2012). In addition, altered CK levels in CKX-overexpressing lines may also directly interfere with ABA homeostasis. There is evidence from Arabidopsis for crosstalk between CK and ABA signaling pathways on the level of hormone biosynthesis and transcriptional regulation (Nishiyama et al., 2011; O'Brien and Benková, 2013). However, the precise regulatory circuits relevant for CK-ABA crosstalk in CKX-transgenic barley remain to be clarified. CKX Expression in Roots Prepares Barley Plant Metabolism for Adverse Growth Conditions Besides an altered ABA content, we observed several other metabolic changes in leaves of transgenic lines that could support an improved tolerance to drought. Drought stress led to an increase in overall amino acid content (Table 1). Several of these amino acids are not only considered as metabolic markers for drought and similar stress conditions but have proven beneficial during stress acclimation or avoidance (Krasensky and Jonak, 2012). Under control conditions, transgenic barley plants displayed several-fold higher concentrations of Pro, Asn, Ser, Thr and GABA. Under drought conditions the content of these compounds increased less strongly in CKX-transgenic barley, indicating - similar to the behavior of ABA - reduced drought sensitivity. Several studies indicated that increased basal levels of certain amino acids might contribute to reduced sensitivity to drought conditions. Analysis of drought-induced changes in the metabolome of two different barley cultivars revealed that the level of Pro was already high in the drought-tolerant cultivar in the absence of drought stress (Chmielewska et al., 2016). The analysis of a diverse population of rice cultivars identified a positive correlation of the levels of Asn, Ser and Thr under control conditions with the performance of the respective cultivar under drought stress (Degenkolbe et al., 2013). In Arabidopsis, higher susceptibility to drought stress correlated with a low content of GABA, and increasing the endogenous GABA level rescued this defect (Mekonnen et al., 2016). It has been argued that increased basal levels of these amino acids represent a biochemical predisposition acting as an efficient drought tolerance mechanism (Chmielewska et al., 2016). As almost nothing is known about links between CK and amino acid metabolism, future studies should aim at understanding the mechanisms underlying the observed metabolic changes in CKX-transgenic barley plants and reveal their functional relevance. Conclusions This study has demonstrated the feasibility of generating cereal plants with a larger root system by increasing CK breakdown in the root, which increases the accumulation of certain elements in leaves and seeds and decreases the sensitivity to drought stress. The ability to 17

18 promote the expansion of the root system in a targeted fashion is an important technological advance. It could, for example, be combined with other approaches, such as the expression of DEEPER ROOTING 1 (DRO1) that impacts the angle of lateral roots and causes roots to grow deeper improving access to water in deep soil layers (Uga et al., 2013). We anticipate that the present approach can be used for other monocot species as well, including wheat and maize, although these have distinct types of roots and root architecture. MATERIALS AND METHODS Plant Materials and Growth Conditions The spring barley cultivar Golden Promise (Forster, 2001) was used for transformation. Transgenic plants were generated using Agrobacterium tumefaciens-mediated transformation of immature embryos (Jacobsen et al., 2006; Horvath et al., 2016). Agrobacterium strain AGL1 containing the binary vector pipkb002 (Himmelbach et al., 2007) was used for all transformations. Determination of the number of T-DNA insertions and identification of homozygous individuals in the T1 progeny (obtained by self-fertilization of regenerated T0 plants) was carried out by quantitative PCR (qpcr) performed on genomic DNA by idna Genetics (Norwich, UK). Unless otherwise stated, plants were grown under controlled greenhouse conditions (20 C/16 C; 16 h/8 h light/dark cycle; 500 μmol m -2 s -1 by metal halide lamps (HQI) supplemented with tungsten bulbs). RNA Isolation and Reverse Transcription Quantitative PCR (RT-qPCR) Analysis Total RNA was extracted from tissues using TRIzol reagent (Invitrogen) following the manufacturer s protocol. RNA was purified using the RNayes MinElute clean up kit (Qiagen). Removal of genomic DNA was achieved using RQ1 RNase-Free DNase (Promega). Two µg of total RNA were used for cdna synthesis using the RevertAid First Strand cdna Synthesis Kit of Fermentas (St. Leon-Rot, Germany) and oligodt-primers. To test cdna yield, qpcr was performed using primers of the barley reference gene ubiquitin-conjugating enzyme (HvUBC10; UniGene Hv.27073). Supplemental Table S5 lists the primer sequences used in the present study. The cdna samples were used to determine gene expression levels by RT-qPCR according to Cortleven et al. (2014). CKX Enzyme Activity Assay The CKX enzyme activity assay was performed by using leaf and root material from threeweek-old soil-grown transgenic plants according to Galuszka et al. (2007). Briefly, samples were powdered in liquid nitrogen and protein was extracted by incubating them in 1.5-fold excess (v/w) of the extraction buffer (0.2 M Tris/HCl, ph 8.0, 0.3% Triton X-100, and 1 mm phenylmethylsulfonylfluoride). For the enzyme activity assay, 2,6-dichlorophenol indophenol 18

19 (DCPIP) was used as the electron acceptor and N6-isopentenyladenine (ip) as substrate. Further quantification of protein content in samples was estimated by using the Bradford method (Bradford, 1976) with bovine serum albumin as the standard. Phytohormone Measurements Quantification of ABA and its degradation products was performed as described in Hosseini et al. (2016). Briefly, frozen leaf material was ground in liquid nitrogen before extraction in ice-cold MeOH:formic acid:water (v/v/v, 15:1:4). ABA, PA and DPA were separated in a UPLC system using a high capacity column (Eclipse Plus C18) using a gradient consisting of 0.1% formic acid and liquid chromatography mass spectrometry (LCMS)-grade methanol. Mass spectrometry was performed using a 6490 triple Quad tandem mass spectrometry (MS-MS) (Agilent, Germany) using [ 2 H6]-ABA as internal standard. For CK analysis 100 mg freeze-dried root material was extracted using methanol/water (1:1, v/v) and purified by solidphase extraction. CKs were eluted with 1 ml 0.35 M ammonia (NH 3 ) dissolved in 60 % MeOH. Dried eluents were re-solved in µl 25% MeOH and quantified by liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) (UPLC-Xevo-TQ-MS, Waters). MS data were processed using TargetLynx V4.1 SCN 904 with internal standard correction. Details of CK extraction, separation, and quantification including external and internal standards has been described in (Eggert and von Wirén, 2017). Quantification of Root System Size and Biomass Barley seeds were germinated on moistened filter paper in the dark for three days and then transferred to light for another two days. Thereafter the seedlings were transferred to a hydroponic system and cultivated for another 15 days. For the hydroponic system 0.1- strength Hoagland solution (1 mm KH 2 PO 4, 0.5 mm KNO 3, 0.4 mm Ca(NO 3 ), 0.2 mm MgSO 4, 0.1 mm FeNaEDTA, 0.01 mm H 3 BO 3, 2 μm MnSO 4, 0.2 μm ZnSO 4, 0.2 μm CuSO 4, 0.1 μm Na 2 MoO 4 and 0.02 mm NaCI) was used (Krämer et al., 1996). The 12 L of nutrient solution per box was properly aerated and changed every 4-5 days. Then, roots and shoots were separated and their fresh weights were determined. Later, samples were dried in an oven at 80 C for 68 hours and the dry weight was recorded. For quantification of root system architecture of soil-grown transgenic plants soil-filled rhizoboxes were used. Rhizoboxes (height 31 cm, width 24 cm, length 32 cm; Schütt Labortechnik GmbH, Germany) were assembled as described by Golldack et al. (2004) and Reibe et al. (2014). The rooting compartment was separated by two pieces of nylon gauze (1μm mesh size; Heidland GmbH & Co. KG, Germany) between two plexiglass frames. In each rhizobox three of these plexiglass frame-nylon gauze constructions were inserted 19

20 (Supplemental Fig. S3D) and pure soil was filled into the rhizoboxes. Eight biological replicates for each genotype were used resulting in eight rhizoboxes. In each rhizobox, three pre-germinated (five-day-old) wild-type and transgenic barley seedlings were inserted between the nylon gauze and grown for 23 days (Supplemental Fig. S3D). After harvest, plants were separated into shoots and roots grown between the nylon gauze. Roots were carefully lifted from within the frames and the gauze and cautiously washed to remove bound soil particles. Before scanning, roots were spread out in a root-positioning tray (20 30 cm) to minimize overlap and scanned with a flatbed scanner (EPSON, EU-88, Japan). Greyscale images obtained in tiff format were analysed with WinRHIZO (Pro Version 2005a; Regent Instruments Inc., Canada). Afterwards, separated shoots and roots were dried at 80 C for 68 hours and the dry weight was recorded. Quantification of Shoot and Seed Element Content Seeds of four independent transgenic lines and the wild type were germinated on filter paper in vitro. Three days after germination (DAG), seedlings were transferred to the greenhouse into an unfertilized (type 0) soil supplied by the company Einheitserde (Sinntal-Altengronau, Germany). Composition of unfertilized soil was tested and certified by Institut Koldingen GmbH (Sarstedt, Germany) as described by Drechsler et al. (2015). Plants were grown for four weeks by supplementing equal amounts of fertilizer solution every second or third day depending on soil moisture. The fertilizer solution was based on the composition of 0.5x Murashige and Skoog (MS) medium containing 10 mm KNO 3 10 mm NaH 2 PO 4, 1 mm MgSO 4, 1 mm CaCl 2, 50 μm Na-Fe-EDTA, 50 μm H 3 BO 3, 50 μm MnSO 4, 18.5 μm ZnSO 4, 50 nm CuSO 4, 50 nm CoCl 2, 0.5 μm NaMoO 4 and 2 mm MES. The solution was adjusted to ph 5.7 with 1 M KOH. Leaf samples from two-month-old plants were dried for 72 h at 65 C, and equal amounts were weighed into polytetrafluoroethylene tubes and digested with a HNO 3 + H 2 O 2 mixture in a pressurized microwave digestion system (MARS from CEM GmbH; Kamp- Lintfort, Germany). The concentrations of macro- and microelements were analyzed by inductively-coupled plasma optical emission spectrometry (ICP-OES, icap 6500 dual OES spectrometer; Thermo Fischer Scientific) with certified standard reference samples as a control. The element content in shoots of drought-stressed plants and from seed samples was determined as outlined above. Growth Conditions for Drought Stress Treatment Seeds of Golden Promise and the transgenic barley lines pepp:ckx2-16 and pepp:ckx2-72 were germinated separately in seed germination trays in a climate-controlled growth chamber for two weeks. Then, germinated seeds were transferred to the cold room for vernalization at 8 C for an additional two weeks. Thereafter, 4-week-old plants were sown in 20

21 L pots filled with 2 kg of peat-based growth substrate in five independent replications. The temperature in the greenhouse was approximately 16 C at night and 20 C during the day with a 16 h/8 h light/dark cycle. The soil moisture content was monitored using the moisture meter HH2 coupled with the soil moisture sensor SM200 (Delta T Devices Ltd., England). Under control condition, the moisture meter device showed 40% soil moisture level corresponding to 100% field capacity (FC). The 10% soil moisture corresponded to 20-25% FC (Lancashire et al., 1991), which causes severe drought stress in barley (Seiler et al, 2011; Hosseini et al. 2016). When the plants had formed between 8-9 tillers watering was stopped for about three days to lower soil moisture to 10%. These drought stress conditions were maintained for two weeks with daily monitoring of soil moisture. Control plants were continuously held at 100% FC. The whole shoots were harvested after two weeks of drought stress for further physiological, biochemical and molecular analysis. Quantification of Photosynthesis-Related Parameters Infra-red gas analysis was carried out on individual fully emerged flag leaves of all three barley lines at 12 days after flowering using a LCpro+ device (ADC Bioscientific Ltd, Great Amwell, England). A constant supply of 400 ppm CO 2 (flow rate 200 μmol s -1 ) was provided by a CO 2 cartridge at a photon flux density of 900 μmol m -2 s -1 by a mixed red/blue LED light source mounted above the leaf chamber head. The net assimilation rate, internal CO 2 concentration, stomatal conductance and transpiration rate were all recorded from five individual plants growing under either well-watered or drought stress conditions, with four technical replications per measurement. All the parameters were recorded in the morning hours starting from 10:00 am to 1:00 pm. The instrument was stabilized for 30 min in the greenhouse where measurements were taken. The measurements were only taken once after the internal CO 2 concentration had stabilized (2-3 min after insertion of the leaf into the measuring chamber). Quantification of Free Amino Acids Free amino acids were extracted as described by Hosseini et al. (2016). Shoots of barley plants were ground in liquid nitrogen and 50 mg of finely powdered fresh material were extracted using 1 ml ice-cold MeOH and chloroform (v/v, 1:1). Fluorescing reagent ACQ (6- aminoquinolyl-n-hydroxysuccinimidylcarbamate) was used for derivatization and detection of amino acids. ACQ was dissolved in 3 mg ml -1 of acetonitrile and incubated at 55 C for 10 min. Then 20 ml of plant extract was derivatized in a cocktail containing 20 µl of ACQ, 160 µl of a 0.2 M boric acid buffer (ph 8.8) in a final volume of 200 µl. The solution was incubated at 55 C for 10 min. The separation of derivatized samples was carried out with a reversed 21

22 phase high-performance liquid chromatography (HPLC) system (Waters, Germany) consisting of a gradient pump (Alliance 2795 HT, Waters, Germany), a degassing module, an autosampler and a 2475 fluorescence detector (Waters). A reversed phase column (XBridge; 150 mm, 5 µm; Waters) was used for separation and detection of amino acids at an excitation wavelength of 300 nm and an emission wavelength of 400 nm. The gradient was accomplished with buffer A containing 140 mm sodium acetate, ph 5.8 (Suprapur, Merck) and 7 mm triethanolamine (Sigma, Germany). Acetonitrile (Roti C Solv HPLC, Roth) and purest HPLC water (Geyer, Germany) were used as eluents B and C. Chromatograms were recorded using the software program Empower Pro (Waters). ACCESSION NUMBERS AtCKX1, AT2G41520; AtCKX2, AT2G19500; EXPRESSED PROTEIN, LOC_Os04g11040; RETROTRANSPOSON PROTEIN, LOC_Os10g31730; PEROXIDASE PROTEIN, LOC_Os03g25330; HvNCED2, AB ; HvABA8'OH, DQ145932; HvP5CS1, AK SUPPLEMEMTAL DATA The following materials are available in the online version of this article. Supplemental Figure S1. Identification and validation of root-specific promoters of rice. Supplemental Figure S2. Validation of root-specific promoters of rice in transgenic Arabidopsis plants. Supplemental Figure S3. Root size of barley plants expressing pepp:ckx. Supplemental Figure S4. Shoot and root size of barley plants expressing pret:ckx or pper:ckx. Supplemental Table S1. Concentration of cytokinin metabolites in roots and shoots of threeweek-old barley plants. Supplemental Table S2. Element concentration in shoots of transgenic barley plants. Supplemental Table S3. Element concentration in shoots of transgenic barley plants grown under well-watered and drought stress conditions. Supplemental Table S4. Element concentration in seeds of transgenic barley plants. Supplemental Table S5. Sequences of primers used in the study. ACKNOWLEDGEMENTS We thank Magdalena Schubert and Hans-Henning Steinbiß for excellent technical support in the production of transgenic plants. We acknowledge funding by the Federal Ministry for 22

23 Education and Research (BMBF) in the frame of the PLANT-KBBE program, project ROOT: Root enhancement for crop improvement

24 TABLES Table 1. Transgenic plants contain higher levels of free amino acids under control conditions. Quantification of free amino acid content was performed using shoot material of wild-type and transgenic plants grown under well-watered and drought stress conditions. Values are means ±SE for each compound and were calculated from a minimum of four to five individual plants per genotype in each treatment. Significant differences were calculated using two-tailed Student s t-test. Bold-face values indicate significant differences (p < 0.05) for the same genotype under the different conditions. Asterisks indicate significant differences compared to wild type (WT) for the same treatment (*, p < 0.05; **, p < 0.01; ***, p < 0.001). FW, fresh weight. Control Drought stress Amino pexp:ckx2 pexp:ckx2 acid (nmol g -1 WT WT #16 #72 #16 #72 FW) His 25 ± ±11** 82 ±5* 245 ± ±5 149 ±10** Asn 127 ± ±133*** 1546 ±182* 3846 ± ± ±280 Ser 127 ± ±85*** 1009 ±46*** 1815 ± ± ±95 Gln 603 ± ± ± ± ± ±117 Gly 41 ± ±17** 134 ±15* 246 ± ±3 245 ±41 Asp 853 ± ± ± ± ± ±42 Glu 808 ± ±310* 2359 ± ± ± ±193 Thr 298 ± ±89* 860 ± ± ± ±81 Ala 240 ± ±72** 1008 ±29* 1331 ± ±45* 1232 ±50 Pro 8 ±2 52 ±7** 43 ±5* 4995 ± ± ±234** Cys 13 ±2 35 ±2*** 43 ±8 34 ±4 26 ±1 27 ±1 Lys 3 ± ±22* 57 ±11* 251 ± ±12* 124 ±21** Tyr 36 ±9 53 ±9 60 ±18 88 ±2 78 ±6 61 ±10 Met 302 ±25 35 ±26*** 10 ±5** 10 ±4 12 ±4 5 ±1 Val 70 ± ±34* 211 ±19* 688 ± ±30 41 ±512* Ile 32 ±22 98 ±13* 73 ±9 315 ± ± ±11* Leu 32 ±24 94 ±18 67 ± ± ± ±7* Phe 31 ±16 91 ±10* 69 ±2 144 ± ±3* 93 ±5* GABA 29 ± ±13* 136 ±24* 187 ± ±12 97 ±5* Total ±2268 ±1158 ±851 ±2091 ±983 ±

25 FIGURE LEGENDS Figure 1. Characterization of transgenic barley lines with root-specific expression of CKX genes. A, Expression of CKX2 under control of the EPP promoter in roots at different developmental stages. B, Expression of CKX1 or CKX2 in roots of different three-week-old transgenic barley lines (T3 generation). For (A) and (B) RT-qPCR was performed using three biological replicates for each line and HvUBC10 as the reference gene. Relative expression of the transgene is shown as 40-ΔCT value, with 26 being the threshold value for expressed genes. Bars represent means ± SD. C, CKX enzyme activity in wild-type (WT) and transgenic lines expressing CKX2. The CKX enzyme activity assay was performed with root extracts of three-week-old soil-grown plants using N 6 -isopentenyladenine (ip) as a substrate. Four biological replicates pooled from 2-3 plants were analyzed for each genotype. Bars represent means ± SE. D, Cytokinin (CK) concentrations in roots of transgenic barley plants compared to WT roots. CK metabolites were quantified in three-week-old plants grown in a hydroponic system. The total content of each major group of CK metabolites in WT was set to 100%. Four biological replicates of 2-3 plants were analyzed for each genotype. Complete data and statistics are shown in Supplemental Table S1. Asterisks indicate statistically significant differences from WT determined using two-tailed Student's t-test (*, p < 0.05; **, p < 0.01). n.d., not detected. Figure 2. Root-specific expression of cytokinin oxidases enhances root system size. A, Root phenotype of two-week-old transgenic lines grown in hydroponic culture. A representative image of individual plants of the wild type (WT) and each transgenic line is shown. Scale bar 2 cm. B, Total root length; C, total root surface area, D, root biomass (dry weight), and E, shoot biomass (dry weight) of two-week-old plants. Total root length and surface area were calculated using the WinRHIZO software. Bars in (B-E) represent means ± SE (n = 20). Asterisks indicate significant differences from the WT as determined by twotailed Student's t-test (*, p < 0.05; **, p < 0.01; ***, p < 0.001). Figure 3. Root-specific expression of cytokinin oxidases does not cause a yield penalty. A, Root and shoot phenotype of 12-week-old plants grown in soil-filled pots. B, Shoot biomass and C, number of tillers of ten-week-old transgenic plants. D, Number of filled spikes, E, total yield per plant, and F, 100-kernel weight of wild-type (WT) and transgenic plants. Bars in B-F represent means ± SE; n = Asterisks indicate significant differences from the WT as determined by two-tailed Student's t-test (* p < 0.01).. Figure 4. Mineral element concentrations in shoots. A, Relative changes in mineral element concentrations in transgenic lines compared to wild type (WT). The concentration of 25

26 each mineral element in WT shoots was set to 100 % and relative differences in transgenic lines are shown in a heat map generated using Multiexperiment Viewer v4.9 (Saeed et al., 2003). The complete data set is shown in Table S2. B, Concentrations of phosphorus (P), sulfur (S), iron (Fe), and zinc (Zn) in eight-week-old soil-grown shoots. Four biological replicates for each genotype were analysed, each containing shoots from 2-3 plants. Bars represent means ± SE. Asterisks indicate significant differences from the WT as determined by two-tailed Student's t-test (*, p < 0.05;**, p < 0.01; ***, p < 0.001). Figure 5. Mineral element concentrations in seeds. A, Relative changes in mineral element concentrations in transgenic lines compared to wild type (WT). The concentration of each mineral element in WT seeds was set to 100% and relative differences in transgenic lines are shown in a heat map generated using Multiexperiment Viewer v4.9 (Saeed et al., 2003). The complete data set is shown in Table S3. B, Concentrations of calcium (Ca), copper (Cu) and zinc (Zn) in seeds of transgenic lines in comparison to WT seeds. Four biological replicates for each genotype were analysed, each containing seeds from 2-3 plants. Bars represent means ± SE. Asterisks indicate significant differences from the WT as determined by two-tailed Student's t-test (*, p < 0.05; **, p < 0.01). Figure 6. Root-specific expression of CKX genes increases tolerance of long-term drought stress. A, Stomatal conductance, B, transpiration rate, C, assimilation rate, D, glucose, E, fructose, and G, sucrose concentration in wild-type (WT) plants and two transgenic lines. Measurements were taken from five biological replicates of each genotype grown under either well-watered (control) or drought-stress conditions, with four technical replications per measurement. Bars represent means ± SE. Asterisks indicate significant differences from the WT as determined by two-tailed Student's t-test (*, p < 0.05; **, p < 0.01; ***, p < 0.001). Differences between control and drought conditions within all genotypes were statistically significant at p < FW, fresh weight. Figure 7. Abscisic acid (ABA) homeostasis and proline (Pro) concentration in pepp:ckx transgenic lines. Leaf concentrations of A, ABA; B, phasic acid (PA); C, dihydrophasic acid (DPA); and D, Pro in wild-type (WT) and transgenic plants grown under control and drought-stress conditions (n = 4-5). E, Relative transcript levels of a gene involved in ABA synthesis (HvNECD2); F, a gene involved in ABA degradation (HvABA-8 - OH); and G, the Pro synthesis gene (HvP5CS1) at the 8-9 tiller stage as determined by RTqPCR. Total RNA was extracted from leaves of WT and transgenic plants grown under control and drought stress conditions. Transcript levels in WT leaves under control conditions was set to 1 (n = 4). Bars represent means ± SE. Asterisks indicate significant differences 26

27 from the WT as determined by two-tailed Student's t-test (*, p < 0.05; **, p < 0.01). Differences between control and drought conditions within all genotypes were statistically significant at p < FW, fresh weight. 27

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36 Figure 1. Characterization of transgenic barley lines with root-specific expression of CKX genes. A, Expression of CKX2 under control of the EPP promoter in roots at different developmental stages. B, Expression of CKX1 or CKX2 in roots of different three-week-old transgenic barley lines (T3 generation). For (A) and (B) RTqPCR was performed using three biological replicates for each line and HvUBC10 as the reference gene. Relative expression of the transgene is shown as 40- CT value, with 26 being the threshold value for expressed genes. Bars represent means ± SD. C, CKX enzyme activity in wild-type (WT) and transgenic lines expressing CKX2. The CKX enzyme activity assay was performed with root extracts of three-week-old soilgrown plants using N 6 -isopentenyladenine (ip) as a substrate. Four biological replicates pooled from 2-3 plants were analyzed for each genotype. Bars represent means ± SE. D, Cytokinin (CK) concentrations in roots of transgenic barley plants compared to WT roots. CK metabolites were quantified in three-week-old plants grown in a hydroponic system. The total content of each major group of CK metabolites in WT was set to 100%. Four biological replicates of 2-3 plants were analyzed for each genotype. Complete data and statistics are shown in Supplemental Table S1. Asterisks indicate statistically significant differences from WT determined using two-tailed Student's t-test (*, p < 0.05; **, p < 0.01). n.d., not detected.

37 Figure 2. Root specific expression of cytokinin oxidases enhances root system size. A, Root phenotype of two-weeks-old transgenic lines grown in hydroponic culture. A, representative image from root systems of individual plants of each line is shown. Scale bar 2 cm. B, Total root length; C, total root surface area, D, root biomass (dry weight), and E, shoot biomass (dry weight) of two-weeks-old plants. Total root length and surface area was calculated using the WinRHIZO TM software. Bars in (B-E) represent means ± SE (n = 20). Asterisks indicate significant differences to the wild type as determined by two-tailed Student's t-test(*,p<0.05;**,p<0.01;***,p< 0.001).

38 Figure 3. Root-specific expression of cytokinin oxidases does not cause a yield penalty. A, Root and shoot phenotype of 12-week-old plants grown in soil-filled pots. B, Shoot biomass and C, number of tillers of ten-week-old transgenic plants. D, Number of filled spikes, E, total yield per plant, and F, 100-kernel weight of wild-type (WT) and transgenic plants. Bars in B-F represent means ± SE; n = Asterisks indicate significant differences from the WT as determined by two-tailed Student's t-test (* p < 0.01 ).ER; Adopted

39 Figure 4. Mineral element concentrations in shoots. A, Relative changes in mineral element concentrations in transgenic lines compared to wild type. The concentration of each mineral element in wild-type shoots was set to 100 % and relative differences in transgenic lines are shown in a heat map generated using Multiexperiment Viewer v4.9 (Saeed et al., 2003). The complete data set is shown in Table S2. B, Concentrations of mineral elements in eight-weeks-old soil-grown shoots. Four biological replicates for each genotype were analysed, each containing shoots from 2-3 plants. Bars represent means ± SE. Asterisks indicate significant differences to the wild type as determined by two-tailed Student's t-test (*, p < 0.05;**, p < 0.01; ***, p < 0.001).

40 Figure 5. Mineral element concentrations in seeds. A, Relative changes in mineral element concentrations in transgenic lines compared to wild type. The concentration of each mineral element in wild-type seeds was set to 100% and relative differences in transgenic lines are shown in a heat map generated using Multiexperiment Viewer v4.9 (Saeed et al., 2003). The complete data set is shown in Table S3. B, Concentrations of calcium (Ca), copper (Cu) and zinc (Zn) in seeds of transgenic lines in comparison to wild-type seeds. Four biological replicates for each genotype were analysed, each containing seeds from 2-3 plants. Bars represent means ± SE. Asterisks indicate significant differences to the wild type as determined by two-tailed Student's t-test (*, p < 0.05; **, p < 0.01).