Driving the expression of RAA1 with a drought-responsive promoter enhances root growth in rice, its accumulation of potassium and its tolerance to moisture stress

Environmental and Experimental Botany 147 (2018) 147–156

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Driving the expression of RAA1 with a drought-responsive promoter enhances root growth in rice, its accumulation of potassium and its tolerance to moisture stress

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Guang Chena,b, , Chaolei Liua, Zhenyu Gaoa, Yu Zhanga, Li Zhua, Jiang Hua, Deyong Rena, ⁎⁎ ⁎ Guohua Xub, , Qian Qiana, a

State Key Laboratory of Rice Biology, China National Rice Research Institute, Hangzhou 310006, PR China State Key Laboratory of Crop Genetics and Germplasm Enhancement, MOA Key Laboratory of Plant Nutrition and Fertilization in Lower-Middle Reaches of the Yangtze River, Nanjing Agricultural University, Nanjing 210095, PR China b

A R T I C L E I N F O

A B S T R A C T

Keywords: Oryza sativa Water deficit RAA1 Root growth Inducible promoter K uptake

Drought impedes the acquisition of potassium (K) by restricting root growth, in turn causing a reduction in the plant's K nutritional status, thereby further depressing its tolerance of the stress. The product of RAA1 (Root Architecture Associated 1) is involved in the auxin-mediated development of the rice root system. Here, the introduction of a transgene comprising RAA1 driven by the promoter of HAK1, a gene which encodes a droughtenhanced K transporter, was shown to exert a positive effect on the size of the root system and the number of adventitious roots formed. Transgenic seedlings demonstrated a higher level of tolerance to moisture stress than wild type (WT) ones, accumulated more K, proline and abscisic acid and suffered a lower level of lipid peroxidation. The genes AKT1, HAK1 and HAK5 were all up-regulated in the roots of transgenic drought-stressed hydroponics-grown seedlings, as were several known stress-responsive genes in the leaves of soil-grown, moisture-stressed transgenic plants. Under moisture deficient conditions, the transgenic plants developed more effective tillers than did WT plants, showed an enhanced level of spikelet fertility and produced larger grains. While under moisture sufficient conditions there was no significant difference in the grain yield of the transgenic and WT lines, under water limiting conditions, the transgenics recorded a 20–40% grain yield advantage over the WT. The implication was that the promotion of root growth and development achieved by enhancing the expression of RAA1 in the root could represent a viable approach for enhancing the productivity of cereal crops exposed to moisture stress.

1. Introduction Drought imposes a major constraint over crop yield. For rice, its most prominent physiological effects are to delay flowering time, to restrict the number of spikelets formed and to reduce the rate of grain filling (Ekanayake et al., 1989; Lee et al., 2017). Plants have evolved a number of molecular, cellular and physiological strategies to either tolerate or avoid moisture stress (Shinozaki and Yamaguchi-Shinozaki, 2007). Genetic engineering may have considerable potential as a means of boosting the naturally occurring levels of stress tolerance (Cui et al., 2016), as exemplified by the positive consequences of heterologously expressing genes encoding a range of either functional or regulatory proteins (Lu et al., 2013; Li et al., 2014a; Cui et al., 2016).

A key adaptation to drought relates to induced changes to the growth and/or development of the root system (Lee et al., 2017). A more robust root system, either in terms of its horizontal or vertical spread, can result in, respectively, a more efficient exploitation of moisture present at normal rooting depth or an improved access to moisture present at greater soil depths (Asch et al., 2005; Lynch, 2013). Enhanced root growth may in principle be achieved by over-expressing one of several genes encoding either functional proteins or transcription factors directly involved in root development: examples of the success of this approach in rice include SGL (Cui et al., 2016), NAC5 (Jeong et al., 2013), NAC6 (Lee et al., 2017), NAC9 (Redillas et al., 2012), NAC10 (Jeong et al., 2010), ERF48 (Jung et al., 2017) and ERF71 (Lee et al., 2016).

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Corresponding authors at: State Key Laboratory of Rice Biology, China National Rice Research Institute, Hangzhou 310006, PR China. Corresponding author: State Key Laboratory of Crop Genetics and Germplasm Enhancement, MOA Key Laboratory of Plant Nutrition and Fertilization in Lower-Middle Reaches of the Yangtze River, Nanjing Agricultural University, Nanjing 210095, PR China. E-mail addresses: [email protected] (G. Chen), [email protected] (G. Xu), [email protected] (Q. Qian). ⁎⁎

https://doi.org/10.1016/j.envexpbot.2017.12.008 Received 8 July 2017; Received in revised form 26 November 2017; Accepted 5 December 2017 Available online 17 December 2017 0098-8472/ © 2017 Elsevier B.V. All rights reserved.

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Fig. 1. The effect of moisture stress on the expression of HAK1. (A) The up-regulation of HAK1 in two week old WT seedlings exposed to moisture stress. (15% PEG for three days). Ubq was the reference sequence for the qRT-PCR assay. (B) Quantification of GUS activity produced by the pHAK1:GUS transgene. (C–L) GUS staining in (C–G) non-stressed and (H-L) PEGstressed transgenic plants. (C,H) Root tip, (D,I) 1 cm proximal to the root tip, (E,J) root lateral branching zone (4 cm proximal from the root tip), (F,K) the root-shoot junction, (G,L) the leaf blade. Bars: 2 mm. Error bars indicate the SE (n = 3). *, **: the mean performance of control and PEG-stressed plants differed significantly from one another at, respectively, P < 0.05 and < 0.01.

regulated gene; it encodes a K transporter protein, which has been predicted to contribute to the maintenance of K homeostasis (Chen et al., 2015b).

Potassium (K), along with nitrogen and phosphorus, is one of the three most important plant nutrients. It affects many aspects of plant performance, including economic yield, resistance to disease and tolerance of abiotic stess (Ahmad et al., 2016b). The K status of a plant has been correlated with its level of hydration and its water use efficiency (Kuchenbuch et al., 1986; Tanguilig et al., 1987). Altering its uptake of K is a major plant response to drought (Andersen et al., 1992; Wang et al., 2004; Mahouachi et al., 2006). An ability to control the loss of K is helpful in the context of supporting osmotic adjustment, sustaining cell expansion, regulating stomatal movement and maintaining photosynthetic activity (Römheld and Kirkby, 2010; Zörb et al., 2014). When drought inhibits the uptake of K by restricting root growth, the resulting reduction in the plant's K status further depresses its tolerance of the stress (Römheld and Kirkby, 2010; Zörb et al., 2014). The putative function of the cluster I–IV KT/HAK/KUP transporters is predicted to contribute to K homeostasis (Chen et al., 2015b). Although two rice K channel proteins (TPKb and AKT1) have been reported to affect tissue K levels and to alter tolerance to osmotic and drought stress (Ahmad et al., 2016a,b), the influence of the KT/KUP/HAK transporters over the plant's tolerance of moisture stress has not been characterized. The constitutive expression of RAA1 (Root Architecture Associated 1) has been shown to increase the production of adventitious roots, but at the same time it also induces a degree of floret sterility (Ge et al., 2004). The aim of the present study was to test the hypothesis that driving RAA1 with either a root-specific or a drought-inducible promoter would retain the favorable effects of its ectopic over-expression, while removing the detrimental effect on floret development. If confirmed, such a transgene construct would represent a potential genetic modificationbased approach for improving the drought tolerance of rice. The promoter chosen here drives HAK1, a K deficiency and moisture stress up-

2. Materials and methods 2.1. Plant growth conditions Following their surface sterilization, grains were germinated in the presence of a nutrient solution (Li et al., 2006). Sets of 20 uniformly sized, one week old seedlings were individually supported over 10 L of nutrient solution; each set of 20 seedlings comprised five WT and 15 transgenic (five seedlings per each of three lines) individuals. The plants were raised under a 14 h photoperiod at a constant temperature of 30 °C during the lit period and of 22 °C during the non-lit period. The relative humidity was maintained at ∼70%. The hydroponic solution was replaced every two days. Moisture stress was imposed on two week old seedlings by introducing 15% (w/v) PEG6000 into the hydroponic solution: this generated a water potential of −0.39 MPa, as measured by a Wescor model 5520 vapor pressure osmometer (www.wescor. com). After one week, the plants were harvested, rinsed for 5 min in 0.1 mM CaSO4, separated into root and shoot material and oven-dried at 105 °C for 30 min and then at 70 °C until a constant weight had been attained. After grinding to a powder, a 50 mg aliquots were digested in 5 mL 18.4 M H2SO4 and 1 mL 9.8 M H2O2 at 270 °C. After cooling, the samples were diluted to 100 mL with distilled water. The concentration of K in the digests was measured using an Optima 2100DV ICP emission spectrometer (PerkinElmer Inc., Shelton, CT, USA), following Chen et al. (2015b). Greenhouse-based pot experiments used soil collected from a China 148

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replaced by the HAK1 promoter. The RAA1 open reading frame was amplified from a cDNA template using the primer pair given in Table S2. The resulting amplicon was digested with BamHI and SpeI and the resulting restriction product ligated into pTCK303. The Ubq promoter was then removed by HindIII/BamHI digestion. The HAK1 promoter was obtained by amplifying the 3010 nt upstream of the HAK1 translation start codon, using the primer pair given in Table S2; after HindIII/BglII digestion, the sequence was ligated to the altered pTCK303 vector using the isocaudamer enzyme ligation method (Chen et al., 2015b). The resulting construct was introduced into Agrobacterium tumefaciens strain EHA105 via electroporation and then transformed into the japonica rice cv. Nipponbare, as described by Chen et al. (2015a). 2.4. Quantification of GUS activity GUS activity was measured in transgenic plants harboring the pHAK1:GUS construct described by Chen et al. (2015b), using the histochemical assay given by Chen et al. (2015a). 2.5. Measurement of root number and length A WinRhizoV4.0b device (Regent Instruments Inc., http://regent.qc. ca) was used to quantify the root system developed by seedlings exposed to the various treatments, following methods described by Chen et al. (2015a). Each measurement was based on the mean performance of five independent plants per line. 2.6. Measurement of relative water content (RWC), rate of water loss and electrolyte leakage RWCs were estimated following Chen et al. (2017). RWC was given by the expression (FW-DW)/(SW-DW) × 100%, where FW represented the leaf fresh weight, DW its dry weight and SW its saturated weight. The rate of water loss from detached leaves was measured following Guo et al. (2016a). The methods used to quantify relative electrolyte leakage have been described by Guo et al. (2016b).

Fig. 2. Characterization of transgenic lines harboring pHAK1:RAA1. (A) Transgene copy number as identified via Southern blot analysis. Genomic DNA of the L1–L7 T1 lines was digested with HindIII and BamHI, and the digests probed with a fragment of the hyg (hygromycin) gene. M: marker, P: positive control. (B) qRT-PCR analysis of RAA1 transcription in the root and shoot of the transgenic lines and WT exposed to PEG stress. The abundance of RAA1 transcript in the roots of non-stressed WT plants was set arbitrarily to 1. Values shown in the form mean ± SE (n = 3). *: the mean performance of WT and the transgenic lines differed significantly from one another at P < 0.05; ns indicates nonsignificant differences.

2.7. Measurement of leaf hydrogen peroxide (H2O2), malondialdehyde (MDA), proline and abscisic acid (ABA) content

National Rice Research Institute experimental farm in Hangzhou, Zhejiang. Each pot was filled with 10 kg air-dried soil containing 164.3 mg/kg exchangeable K, as determined by extraction in 1 M neutral ammonium acetate (Chen et al., 2015a). Following the Karaba et al. (2007) procedure, five week old rice plants were exposed to a three week period of limited water supply (40% field capacity), maintaining a control set of fully watered plants. A similar level of moisture stress was also applied to pot-grown plants from the booting stage to plant maturity; once again a control set of plants was kept fully watered. Each treatment was replicated five times.

The H2O2 content of leaf samples was determined following their equilibration in 5.3 mM TiCl4 dissolved in 3.7 M H2SO4, based on the procedure given by Mostofa and Fujita (2013). The leaf MDA content was assessed using a protocol given by Chen et al., (2017). The leaf proline content was determined following the Bates et al. (1973) procedure. The leaf ABA content was assessed following Cai et al. (2015). 2.8. Southern blotting The presence and copy number of the pHAK1:RAA1 transgene in T1 plants were evaluated using Southern blotting, following procedures described by Chen et al. (2015a).

2.2. Quantitative real time PCR (qRT-PCR) The procedures used to conduct qRT-PCR followed those given by Chen et al. (2015a). RNA was extracted from the entire root and shoot of both hydroponically raised WT and transgenic seedlings subjected to PEG treatment, and from the youngest two leaves of soil-grown plants. The rice Ubq (LOC_Os03g13170) gene was chosen as the reference sequence, and relative transcript abundances were calculated following the suggestion of Li et al. (2014b). The sequences of the various primers used are listed in Table S1.

2.9. Statistical analysis Analyses of variance were carried out using routines implemented in SPSS v10 software (SPSS Inc., Chicago, IL, USA). 3. Results 3.1. Moisture stress enhanced HAK1 expression, but decreased the abundance of RAA1 transcript in both the root and shoot

2.3. Construction of the pHAK1:RAA1 transgene and its introduction into rice

As envisaged by qRT-PCR, the transcription of HAK1 in the root appeared to be some four fold higher in plants subjected to moisture stress than in non-stressed ones, and two fold higher in the shoot

The Ubq promoter present in pTCK303 (Chen et al., 2015a) was 149

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Fig. 3. Seedling growth of pHAK1:RAA1 transgenics in response to moisture stress. (A,B) The appearance of WT and L1–L3 seedlings (A) under non-stressed conditions, (B) when exposed to 15% PEG. White bars: 5 cm, yellow bars: 1 cm. (C,D) The accumulation of biomass by (C) the root, (D) the shoot. Values shown in the form mean ± SE (n = 5). *: the mean performance of WT and the transgenic lines differed significantly from one another at P < 0.05; ns indicates non-significant differences. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 4. The development of the root system of pHAK1:RAA1 transgenics and WT in response to moisture stress. Plants grown under (A) non-stressed conditions and (B) in a medium containing 15% PEG. Bars: 5 cm. (C) Adventitious root number, (D) adventitious root length, (E) root surface area and (F) total root length. Values shown in the form mean ± SE (n = 5). *: the mean performance of WT and transgenic plants differed significantly from one another at P < 0.05; ns indicates non-significant differences.

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Fig. 5. The accumulation of K in WT and transgenic seedlings harboring pHAK1:RAA1 in response to moisture stress. The K content of (A) the root and (B) the shoot. Values shown in the form mean ± SE (n = 5). *: the mean performance of WT and transgenic plants differed significantly from one another at P < 0.05; ns indicates non-significant differences. DW: dry weight.

stressed L1–L3 seedlings averaged about 25% higher than that recorded by WT seedlings (Fig. 3C,D). PEG-stressed WT plants developed markedly fewer adventitious roots, and those which did emerge tended to be shorter than those developed by non-stressed seedlings (Fig. 4A–C); in addition, both the surface area and length of the roots were negatively affected (Fig. 4D–F). In contrast, L1–L3 seedlings responded positively to the treatment (Fig. 4C–F). Analogous behavior was expressed consistently by the T0 (data not shown), T1 (Fig. S3) and T2 (Fig. 4) plants.

(Fig. 1A). The inducibility of the HAK1 promoter by drought was confirmed by characterizing the effect of introducing a transgene comprising GUS fused to the 3010 nt sequence upstream of the HAK1 transcription start codon (Chen et al., 2015b). When cultured for seven days in the presence of 15% PEG, GUS activity in the transgenic plants' roots was increased by two fold over the level observed under nonstressed condition, and by 1.5 fold in their shoots (Fig. 1B). A histological analysis indicated that the HAK1 promoter was active throughout both primary (Figs. 1C, D, H, I and S1) and lateral (Figs. 1E, J and S1) roots, as well as moderately so in the leaf blades (Figs. 1G, L and S1). The abundance of RAA1 transcript was overall greater in the root than in the shoot tissue, whether or not the plants experienced moisture stress. The gene’s involvement in the root growth of moisture-stressed plants was implied by its suppression in both the root and shoot of plants challenged by PEG stress (Fig. S2).

3.4. The effect of the pHAK1:RAA1 transgene on seedling K uptake There was no significant difference between the amount of K accumulated by hydroponically grown WT and L1–L3 seedlings in the absence of PEG; however in its presence, the latters’ roots and shoots contained, respectively, 40% and 35% more K than did those of WT (Fig. 5A,B). When qRT-PCR was used to assess the transcriptional activity of genes encoding Ktransport proteins, it was clear that the PEG treatment up-regulated AKT1 (encoding a K channel protein) and both HAK1 and HAK5 (K transporters) in the roots (Fig. 6A–C). The extent of the up-regulation was stronger in L1-L3 than in WT (Fig. 6).

3.2. Transgenic rice plants harboring the pHAK1:RAA1 transgene In all, 28 independent putative T0 selections were validated by Southern hybridization as harboring the pHAK1:RAA1 transgene. The appearance of the roots of one of these lines and of its T1 segregants lacking the transgene is illustrated in Fig. S3A and S3B. Since there was no significant difference between the null segregants and WT under both the stressed and non-stressed growing conditions in terms of either the abundance of RAA1 transcript or root growth (the number of adventitious roots formed, overall length and biomass) (Fig. S3C-F), WT plants were used as the negative control in subsequent experiments based on T2 lines. Of the set of confirmed T1 transgenics, a subset of seven presented a comparable root architecture and grain yield when the plants were subjected to moisture stress (data not shown); of these, three single copy transformants (L1–L3) (Fig. 2A) were retained for further analysis. Stress-challenged L1–L3 plants accumulated two to three times as much RAA1 transcript in their roots than did the roots of non-stressed plants, while in WT plants, the stress treatment markedly suppressed the transcription of RAA1 (Fig. 2B). Although RAA1 was up-regulated by moisture stress in the transgenic plants, its transcript abundance in the shoot remained relatively low (Fig. 2B).

3.5. The effect of the pHAK1:RAA1 transgene on the growth and K uptake of soil-grown plants The tolerance of the pHAK1:RAA1 transgenics, both in the T1 and T2 generations, was tested in soil-grown plants. Moisture stress was applied at two physiological stages to pot-grown WT plants, T1 segregants lacking the transgene (“null segregants”) and T1 segregants retaining the transgene (“transgenic segregants”). For the first set, the stress was imposed at the tillering stage, while for the second it was imposed at the booting stage. The stressed transgenic segregants accumulated significantly more root and shoot biomass than did either the WT or null segregants, and produced a higher grain yield per plant (data not shown). T2 generation and WT plants were similarly tested for their drought tolerance in a pot trial. Prior to the imposition of the stress, the growth of the transgenic and WT plants was similar, but in response to the stress, the former produced a higher root and shoot biomass (Fig. S4A,B). As was also observed in the hydroponics experiment, the stress promoted root growth in the transgenic plants, but suppressed it in the WT ones (Fig. S4A). The shoot biomass of the moisture-stressed L1–L3 plants was significantly greater than that achieved by WT plants (Fig. S4B). There was no significant difference in the tissue K content of nonstressed transgenic and WT plants (Fig. S4C,D), but in response to moisture stress, the transgenic plants accumulated respectively ∼50% and ∼45% more K in their roots and shoots than was managed by WT plants.

3.3. The effect of the pHAK1:RAA1 transgene on seedling growth and root architecture When the performance of hydroponically grown WT and line L1–L3 plants was compared, no difference in seedling growth was observed in the absence of PEG stress (Fig. 3A). However, in its presence, the WT seedlings suffered from wilting and leaf chlorosis, symptoms which were not apparent in the L1–L3 plants (Fig. 3B). The stress treatment suppressed root growth in WT seedlings, which was not the case for the transgenics; consequently, the latter were able to develop a more extensive root system (Fig. 3B,C). The stress-induced reduction in shoot growth was less severe for the transgenic seedlings than for the WT ones (Fig. 3B,D). The total biomass (root + shoot) accumulated by the PEG151

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tissue H2O2 content revealed that the latter were less responsive to moisture stress than the former, although there was no discernible difference between the lines in the absence of stress (Fig. 8A). Measurement of electrolyte leakage indicated that the WT and L1–L3 plants were indistinguishable in the absence of moisture stress, but in its presence, leakage was considerably higher from WT than from transgenic leaf samples (Fig. 8B). Similarly, with respect to tissue MDA content, moisture-stressed transgenic plants proved superior to stressed WT ones (Fig. 8C). When the accumulation of proline was compared, non-stressed transgenic and WT plants behaved similarly; but under moisture stress, the induced increase was 30% greater in the transgenic plants than in the WT ones (Fig. 8D). 3.7. The expression of pHAK1:RAA1 altered ABA content and the abundance of stress-responsive gene transcripts in moisture-stressed plants ABA controls many aspects of the plant response to drought. As plants expressing the pHAK1:RAA1 transgene displayed an enhanced drought tolerance, it was interest to compare the ABA content of the leaf of the WT and transgenic plants. No significant difference was detected between the lines raised under non-stressed conditions (Fig. 9A). Moisture stress induced an increase in the leaf ABA content of both the transgenic and WT plants, but the transgenic leaves accumulated some 28% more than the WT ones. When the effect of expressing the pHAK1:RAA1 trangene on the transcription of four stress-related genes (P5CS1 (Sripinyowanich et al., 2013), SNAC1 (Hu et al., 2006), DREB1A (Dubouzet et al., 2003) and DREB2B (Chen et al., 2008)) was analyzed, under non-stressed conditions, they all proved to be weakly transcribed, with no perceptible effect of the presence of the transgene; in the moisture-stressed plants, however, all four were substantially upregulated (Fig. 9B–E). The effect was particularly strong for P5CS1 (Fig. 9B), consistent with the transgenic lines' greater propensity to accumulate proline (Fig. 8D). The relative increase in SNAC1, DREB1A and DREB2B transcript abundance experienced by the L1-L3 plants compared to the WT ones lay in the range 0.50–1.5 fold (Fig. 9C–E). 3.8. The expression of pHAK1:RAA1 significantly improved both drought tolerance and grain yield An agronomic comparison between the transgenic and WT plants was based on a trial in which the soil moisture content was controlled. Under well irrigated conditions, the performance of the transgenic lines did not differ from that of WT; however, when the moisture remained below 40% field capacity between the booting stage and physiological maturity, the transgenic plants produced a 15–25% higher number of effective tillers (Fig. 10A), enjoyed a 13–22% higher level of spikelet fertility (Fig. 10B), set grain having a 10–20% higher thousand grain weight (Fig. 10C) and yielded 20–40% more grain per plant (Fig. 10D). 4. Discussion

Fig. 6. Transcript abundance of AKT1 and the HAK genes in WT and transgenic seedlings harboring pHAK1:RAA1 in response to moisture stress. The qRT-PCR signals were normalized against the abundance of the reference gene (Ubq) transcripts. Values shown in the form mean ± SE (n = 3). *: the mean performance of WT and transgenic plants differed significantly from one another at P < 0.05; ns indicates non-significant differences.

4.1. The greater drought tolerance resulting from the expression of the pHAK1:RAA1 transgene reflects its effect on a number of traits Moisture stress is one of the leading constraints over crop productivity (Lobell et al., 2014), and will become more prevalent if the consensus prediction of future global warming proves correct (Ahuja et al., 2010). Thus, crop science will need to prioritize gaining as full an understanding as possible of the mechanistic basis of drought tolerance in order to elucidate breeding strategies targeting the mitigation of losses caused by this stress. Here, it was demonstrated that rice plants harboring the pHAK1:RAA1 transgene were able to better tolerate drought than WT ones. The enhanced tolerance exhibited by the transgenic plants represented the consequences of alterations at the morphological, physiological and molecular levels. Their ability to form a more extensive

3.6. The effect of the pHAK1:RAA1 transgene on the water retention and the occurrence of lipid peroxidation in soil-grown plants A leaf’s RWC and its rate of water loss represent measures of a plant’s capacity to retain water. The impact of moisture stress was to reduce the leaf RWC in both WT and transgenic plants, but the parameter remained significantly higher in the latter plants (Fig. 7A). Water loss from detached leaves occurred more rapidly from WT leaves than from transgenic ones (Fig. 7B). A comparison between the WT and transgenic plants with respect to 152

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Fig. 7. The expression of the pHAK1:RAA1 transgene enhanced the ability of soil-grown plants to acquire and retain water under moisture-stressed conditions. A set of five week old rice plants was exposed to a three week period of limited water supply (40% field capacity), while a control set was kept at 100% field capacity. Measurements were taken of (A) relative water content, (B) the rate of water loss from a detached leaf, sampled at a range of time points. Values shown in the form mean ± SE (n = 5). *: the mean performance of WT and transgenic plants differed significantly from one another at P < 0.05; ns indicates non-significant differences.

Fig. 8. Lipid peroxidation in the leaf of WT and transgenic lines harboring pHAK1:RAA1 in response to moisture stress. (A-C) Physiological indices reflecting lipid peroxidation, namely: (A) H2O2 content, (B) relative electrolyte leakage, (C) MDA content. (D) proline content. Values shown in the form mean ± SE (n = 5). *: the mean performance of WT and transgenic plants differed significantly from one another at P < 0.05; ns indicates non-significant differences. FW: fresh weight.

proline synthesis and is one of the enzymes most closely associated with abiotic stress tolerance (Igarashi et al., 1997; Zhu et al., 1998). The markedly higher abundance of P5CS1 transcript seen in the moisturestressed transgenic plants was consistent with their enhanced proline content (Figs. 8D, 9A). An increase in proline content would be expected to generate a higher level of osmolality, thereby rendering the plants more effective with respect to water retention (Song et al., 2012). As a result, the transgenic plants’ leaves displayed a higher RWC and suffered a lower rate of water loss than was the case for those developed by WT plants (Fig. 7). Since proline can also mitigate against oxidative damage during a stress episode (Székely et al., 2008), its enhancement could also have contributed to the transgenics’ leaves suffering less from electrolyte loss and accumulating less MDA in response to moisture stress (Fig. 8B–D). The increased leaf ABA content achieved by expressing pHAK1:RAA1 (Fig. 9A) provided a further indication of an improved moisture stress tolerance. Generally, a high level of endogenous ABA might strengthen and/or accelerate stress response and thus is thought to support the plant's tolerance of biotic stress tolerance. For example, a loss-of-function mutation in the rice gene OsDSM2/ OsBCH1, encoding one of three putative β-carotene hydroxylases involved in the synthesis of the ABA precursor zeaxanthin, results in a reduction in leaf ABA content and a weakened tolerance to drought (Du et al., 2010); in contrast, the over-expression of Os03g04070, encoding a stress-responsive NAC transcription factor, boosts leaf ABA content and strengthens drought tolerance (Hong et al., 2016).

root system in itself should contribute to their capacity to extract water from a moisture deficient soil. Altering the architecture of the root system can have a positive impact on crop drought tolerance (Cui et al., 2016), and this trait is influenced by a number of transcription factors (Yu et al., 2013; Cheng et al., 2016; Jung et al., 2017; Lee et al., 2016, 2017). The coupling of RAA1 to the HAK1 promoter was designed to trigger its expression under conditions of moisture stress. Dependent on the growth medium and the stress agent employed (hydroponics vs soil, water withholding vs PEG), the expression of the transgene increased root biomass by some 50–70% under moisture deficient conditions (Figs. 3C, S4A). The presence of the transgene was also associated with a lowered tissue content of both H2O2 and MDA (Fig. 8A,C), along with an enhanced accumulation of the osmoprotectant proline (Fig. 8D). Droughtstressed rice is known to accumulate H2O2 (Chen et al., 2017; Jiang et al., 2016), while MDA has been widely used as a proxy for oxidative stress-induced lipid peroxidation (Zhao et al., 2014). In accordance with the reduced production of MDA in the transgenic plants, the extent of electrolyte leakage was lower than from WT tissue (Fig. 8B). The strong implication is that membrane integrity was better conserved in the transgenic plants. Proline is generally considered to act as an osmoprotectant in stressed plants (Xiang et al., 2007; Zhao et al., 2014). In rice, evidence has been presented suggesting that it supports osmotic adjustment under conditions of moisture stress (Chen et al., 2017; Hong et al., 2016; Jiang et al., 2016). The product of P5CS1 is a catalyst for 153

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Fig. 9. The effect of pHAK1:RAA1 expression on leaf ABA content and the level of transcription of selected stress-related genes. The presence of the transgene (A) enhanced the accumulation of ABA and (B-E) upregulated each of the stress-related genes (B) OsP5CS1, (C) OsSNAC1, (D) OsDREB1A, (E) OsDREB2B. The qRT-PCR outputs in (B) through (E) were normalized against those targeted at the reference gene Ubq. Data are shown in the form mean ± SE (n = 3). *: the mean performance of WT and transgenic plants differed significantly from one another at P < 0.05; ns indicates non-significant differences. FW: fresh weight.

Fig. 10. Transgenic lines harboring pHAK1:RAA1 exhibited an improved tolerance to moisture stress at the reproductive stage. Moisture stress was applied to pot-grown plants at the booting stage, by maintaining the soil water content at 40% of field capacity. (A) Effective tiller number per plant, (B) % grain set, (C) one thousand grain weight, (D) grain yield per plant. Values shown in the form mean ± SE (n = 5). *: the mean performance of WT and transgenic plants differed significantly from one another at P < 0.05; ns indicates non-significant differences.

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et al., 2014). The indications are therefore that the ectopic expression of RAA1 expanded the growth of the roots, thereby boosting the accumulation of K, and potentially up-regulating genes encoding K transporters/channels. Increasing the uptake of K by manipulating RAA1 expression successfully improved the growth and grain yield of moisture-stressed plants (Figs. 10 and S4 A,B).

At the molecular level, a correlation was established between the transcriptional response and stress tolerance. The transcription profiling of four stress-related genes showed that they were all up-regulated in stressed plants harboring pHAK1:RAA1 (Fig. 9). P5CS1 encodes a key enzyme responsible for proline accumulation (Yoshiba et al., 1999). SNAC1 enhances drought tolerance in transgenic rice experiencing severe moisture stress during both the vegetative and reproductive stages (Hu et al., 2006). The over-expression of DREB1A in Arabidopsis thaliana results in plants exhibiting a heightened tolerance to moisture stress (Dubouzet et al., 2003). The constitutive expression of DREB2B in rice significantly improves its tolerance of moisture deficit (Chen et al., 2008). The implication is that it is the induction of these (and possibly other) stress-related genes which underpins the beneficial effect of the pHAK1:RAA1 transgene on the drought tolerance of rice. Additionally, the induction of stress/ABA-responsive genes in pHAK1:RAA1 transgene carriers may contribute to the increased accumulation of ABA under moisture stress conditions. Many studies have reported how increases in ABA content initiate ABA-mediated pathways regulating the expression of stress-responsive genes (Xiong et al., 2002; Cai et al., 2015; Hong et al., 2016). Overall, the present data imply that the expression under moisture-stressed conditions of the pHAK1:RAA1 transgene promotes ABA synthesis and thereby, through the induction of stress-responsive genes, improves the plant's level of tolerance to drought.

4.3. The use of a drought-inducible promoter mitigated the detrimental effects of ectopic RAA1 expression on floret development The constitutive expression of a number of transcription factors can generate undesirable phenotypes (Kasuga et al., 1999; Zhao et al., 2009; Morran et al., 2011). A strategy to avoid aspects of this problem is to substitute a stress-inducible promoter for the more usually employed constitutive one (Kasuga et al., 1999; Morran et al., 2011; Kovalchuk et al., 2013). The choice here of promoter to drive RAA1 without inducing its undesirable effect on the floret was based on the upstream sequence of HAK1, a gene induced in the rice root by osmotic/drought stress (Fig. 1); its product co-localizes with RAA1 (Ge et al., 2004; Chen et al., 2015b). When RAA1 was placed under the control of the HAK1 promoter, both the leaf and floret phenotypes were indistinguishable from those in WT, whether or not the plants were subjected to moisture stress. The inference is that the basal activity of HAK1 in the shoot is insufficient to generate an undesirable titer of RAA1 transcript. A range of promoters should be available in order to drive RAA1 expression in the appropriate time and place, resulting in a choice of transgene constructs able to enhance the tolerance of rice (and other cereal species) to osmotic and/or moisture stress.

4.2. The enhanced expression of RAA1 driven by the HAK1 promoter increased K accumulation, thereby boosting moisture stress tolerance The K status of a plant is closely correlated with its tissue water content and its efficiency of water use (Kuchenbuch et al., 1986; Tanguilig et al., 1987; Ahmad et al., 2016a). A rise in K uptake is one of the primary responses of plants exposed to drought (Andersen et al., 1992; Wang et al., 2004; Ahmad et al., 2016b). Limiting the loss of K has a positive effect on water retention, ensures the appropriate regulation of stomatal movement and helps maintain photosynthetic activity (Römheld and Kirkby, 2010; Zörb et al., 2014). In the presence of moisture stress, tissue K contents were markedly higher in the pHAK1:RAA1 transgenics than in WT plants (Figs. 5A,B and S4C,D). The more extensive root system formed by the transgenic plants doubtless contributed to this, as the acquisition of nutrients responds to root surface area. In an A. thaliana mutant carrying a T-DNA insertion in the HAK homolog TRH1 (AtKUP4/AtKT3), both the uptake of Rb+ and root hair growth were inhibited, indicating that K uptake is specifically required for root hair cell elongation (Rigas et al., 2001). Following the imposition of moisture stress, the transgenics harboring pHAK1:RAA1 took up 65–75% more K than did WT plants in the hydroponics assay, and 80–90% more in soil-grown materials (Figs. 5A,B; S4C,D). The improvement with respect to K uptake and the enlarged root growth were closely correlated: the presence of pHAK1:RAA1 raised root biomass by 45–55% in the hydroponics assay, and 60–80% in soil-grown plants (Figs. 3C; S4A). The more extensive root system induced by the expression of pHAK1:RAA1 probably contributes to their more effective acquisition of K, thereby equipping them with greater tolerance to moisture stress. When the pHAK1:RAA1 transgenic lines were challenged with moisture stress, the transcription of each of AKT1, HAK1 and HAK5 was at a higher level than in equivalent WT plants (Fig. 6A–C). These changes could be in part due to alterations in the plant's K status, brought about by the reprogramming of root growth induced by the expression of pHAK1:RAA1 (Figs. 5A,B; S4C,D). The constitutive expression of AKT1 in rice is known to heighten tissue K content, especially in the root (Ahmad et al., 2016b). HAK1 is essential for maintaining K mediated-growth and is important for the process of both K uptake and transportation (Chen et al., 2015b); meanwhile HAK5 contributes to K uptake into roots experiencing a low external K concentration and in the translocation of K from the root to the shoot (Yang

5. Conclusion The present experiments have demonstrated that expressing RAA1 under the control of the HAK1 promoter significantly increased the tolerance of rice to moisture stress, by altering the architecture of the root system, the plant's ability to take up K and to accumulate proline and ABA; it also reduced the peroxidation of the plasma membrane and up-regulated several known stress-related genes. Unlike transgenic plants in which RAA1 is constitutively expressed, the pHAK1:RAA1 transgene had no visible deleterious effect on the reproductive structures of the plant, and growth appeared normal whether the plants were grown in soil or hydroponically. The implication is that driving RAA1 with a drought-inducible promoter (such as pHAK1) represents a means to improve the level of tolerance of moisture stress in both rice and other cereal species. Since HAK1 is induced by both moisture stress (as shown here) and by K deficiency (Chen et al., 2015b), it would be worthwhile testing whether plants harboring pHAK1:RAA1 are able to elaborate larger root systems and display an improved ability to take up K under conditions where both stresses are present. These traits would be expected to have a positive effect on grain yield in crops grown under moisture-limited conditions in soils which are deficient for K. Acknowledgments This work was funded by National Natural Science Foundation of China (No.31601811) and the Central Level Scientific Research Institutes for the Basic Research and Development Special Fund Business (Grant No. 2015RG001-2). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at https://doi.org/10.1016/j.envexpbot.2017.12.008. References Ahmad, I., Devonshire, J., Mohamed, R., Schultze, M., Maathuis, F.J., 2016a.

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