Characteristics of transgenic plants compared to wild-type upon drought stress 

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Rice root anatomy

The radial anatomy of rice roots is typical for semiaquatic plants (Figure 4). Though having various size or number of cell layers in each tissue, in general, primary roots and large lateral roots include epidermis, exodermis, sclerenchyma, mesodermis/ aerenchyma, endodermis and center cylinder form the outer to the inner. Small lateral roots display much simpler internal structure with no mesodermis and aerenchyma. Each of the epidermis, exodermis, sclerenchyma or endodermis contains one single cell layer. Sclerenchyma is highly lignified but not composed of suberin, it can be a complement structure against the weakness of extensive formation of aerenchyma and limits oxygen loss from the root (Ranathunge et al., 2003). Different from other tissues, mesodermis consists of several cell layers. At mature zone of roots, mesodermis differentiates into spokes and aerenchyma, which acts as an oxygen reservoir for rice to grow in flooding condition. Aerenchyma starts being established at about 1-2 cm and completed at 10 cm from the root apex (Ranathunge et al., 2003, 2004). Casparian strips development and suberin lamellae deposition are two typical events occurring in the cell wall of exodermis and endodermis. Casparian strips deposit on the radial and transverse walls of the cells, whereas, suberin lamellae develop in inner tangential walls (Figure 5) (Clark and Harris, 1981). Casparian strips are strongly lignified (Schreiber, 1996). Suberin is a biopolymer consisting of aliphatic and aromatic domains. Casparian strips develop sooner compared to the suberin lamellae which commences at about 2 cm and is well-developed at 10 cm from the root apex in endodermis. Casparian strips develop later in exodermis, about 3 cm from the root tip. Suberin deposition in endodermal cell walls starts at about 2 cm, matures at 5-7 cm and completes at about 10 cm from the root tip with its thickness increasing along root towards the root base. Exodermal suberin also develops a bit later at about 3 cm from the root apex (Ranathunge et al., 2003, 2004; Schreiber et al., 2005).

Rice AQP expression in normal conditions

Reverse transcription-PCR (RT-PCR) was a technique among others applied to estimate rice AQP transcript abundance (Sakurai et al., 2005; Guo et al., 2006; Sakurai-Ishikawa et al., 2011; Grondin et al., 2016). Additionally, isoform-specific antibodies were developed for immuno-chemistry applications (Sakurai et al., 2008; Sakurai-Ishikawa et al., 2011).

Tissue-specific expression

In rice, as in other plant species, AQPs were reported to be organ-specifically expressed at different levels, depending on growth stage, and variety (Table 3). For instance, in cv. Akitakomachi, several AQPs were expressed predominantly in root system such as OsPIP1;3, OsPIP2;3, OsPIP2;4, OsPIP2;5, OsTIP2;1 and OsNIP2;1, while others were more expressed in leaf blades including OsPIP2;7, OsPIP2;8, OsTIP1;2, OsTIP3;1, OsTIP3;2, OsTIP4;2, OsTIP4;3, OsTIP5;1, OsNIP1;1, OsNIP1;2, OsNIP1;4, OsNIP3;2, OsNIP3;3 and OsNIP4;1 (Sakurai et al., 2005, 2008). Interestingly, eight genes (OsPIP1;1, OsPIP1;2, OsPIP2;1, OsPIP2;2, OsPIP2;6, OsTIP2;2, OsTIP4;1 and OsSIP1;1) were detected almost equally in both roots and leaf blades (Sakurai et al., 2005, 2008; Sakurai-Ishikawa et al., 2011). In contrast, OsPIP2;1 and OsPIP2;4 were not detected in cv. Moroberekan roots, neither OsPIP2;4 in cv. Azucena roots (Grondin et al., 2016), while this latter had the highest transcript level in cv. Zhonghua 11 roots (Guo et al., 2006). OsPIP2;1, OsPIP2;2, OsPIP2;6, OsPIP2;7 and OsTIP4;1 could not be detected in roots of Giza 178, Sakha 101, IR64 and PLS2 cv. (Nada and Abogadallah, 2014). Moroberekan, a moderate drought tolerance rice cv., showed higher relative transcript abundance of OsPIP1;1, OsPIP1;2, OsPIP2;2, OsPIP2;6 and OsPIP2;8 in roots compared to the drought susceptible IR64 (Grondin et al., 2016).

Role of AQPs in the response of rice to drought and salt stress

As in other plant species, many studies reported that in rice, AQP expression varied according to stimuli of the environment. For instance, exposure of roots to low temperature for a long period (2–5 days) induced a compensatory increase in root hydraulic properties of rice root system, correlated with enhanced expression of OsPIP2;5 in root. Since shoot has to be maintained in its integrity at control temperature, it was suggested the involvement of a shoot-to-root signal (Ahamed et al., 2012). Importantly, in agreement with the role of AQPs in osmoregulation, effects of water and salt stresses were mostly reported (Maurel et al., 2015).

Inhibition of root hydraulic properties upon drought and salt stress

Because osmotic stress reduces water uptake, plant should balance the situation by enhancing root hydraulic conductivity (Lpr). However, reported data indicated different strategies. Hence, the contribution of AQPs, represented by the percentage of Lpr inhibition by azide (NaN3), in three moderate drought tolerance rice cultivars, Azucena, Moroberekan and Dular, was either significantly decreased, not changed or significantly increased by drought stress, respectively (Grondin et al., 2016). Under salt treatment, Lpr of Azucena and Bala rice cultivars were reported to significantly be reduced (Meng and Fricke, 2017). Reduction of Lpr under salt stress was also reported in other plant species, such as Arabidopsis (Boursiac et al., 2005), and barley (Horie et al., 2011); the Lpr reduction exhibiting a shutdown of water transport to minimize water loss (Horie et al., 2011). Since AQPs mediate water transport and are major components of Lpr, their regulation may reflect a strategy of plants in response to drought and salt stress.

Regulation of AQP expression in rice upon drought and salt stress

A likely number of articles studied the regulation of rice AQPs under drought (osmotic) and salt stress at mRNA level. There was a large variation of expression patterns of particular AQP genes, it may be attributed to differences in cultivars, growth stages, level and duration of treatments. Nevertheless, the results suggested a functional coordination between OsPIPs and OsTIPs in water deficit and salt stress.
Many OsPIPs and OsTIPs, analyzed at transcript abundance level, were reported to be upregulated in both leaves and roots of rice grown in low air humidity (Kuwagata et al., 2012). In response to drought stress, mimicked by polyethylene glycol (PEG) treatment, in 29 leaves, most of OsPIP gene expression was not altered or down-regulated; only the expression of OsPIP1;2, OsPIP1;3 and OsPIP2;3 was up-regulated by 6 h of treatment in some cultivars. Meanwhile, the expression of OsTIPs (OsTIP1;1, OsTIP1;2, OsTIP2;2 and OsTIP4;1, OsTIP4;2 ) was up-regulated and peaked at 4-8 h, then down regulated at 10 h; OsTIP4;3 expression was slightly up-regulated. However, in roots, the expression of many PIP (such as OsPIP1;3, OsPIP2;1, OsPIP2;5 and OsPIP2;7) and TIP (such as OsTIP1;1, OsTIP1;2, OsTIP4;1 and OsTIP4;2) genes was up-regulated upon dehydration stress (Liu et al., 1994; Lian et al., 2004, 2006; Guo et al., 2006; Li et al., 2008; Henry et al., 2012). Dry-down soil experiments showed the upregulation of mRNA levels of almost all PIPs and TIPs in leaves, with a higher effect in indica compared to japonica rice, whereas down-regulations of expression were observed in roots (Nada and Abogadallah, 2014; Grondin et al., 2016).
In response to salt stress, OsPIPs transcripts were decreased in leaves in the first 2 h then increased and reached a peak at 6 h of treatment. Differently in roots, OsPIP2 transcripts were not changed or even slightly decreased during one day of salt stress; while OsPIP1 transcripts showed an increase, especially at the first 2 or 6 h (Guo et al., 2006). However, almost all OsTIP transcripts were upregulated, except OsTIP2;2 and OSTIP4;3 in both leaves and roots (Liu et al., 1994; Li et al., 2008).
These data obtained on the transcript accumulation have to be balanced with their absence or weak relationship with rice root hydraulic properties. Therefore, it is difficult to identify a steady pattern for the role of AQPs in response to abiotic stress based on transcriptomic data (Grondin et al., 2016; Meng and Fricke, 2017). Analysis at protein level may reflect more reliably the functional activity of AQPs in response to stress. Thus, study in parallel with two rice cultivars Puluik Arang and Akitakomachi, in which the former is the most drought tolerant, showed that Puluik Arang had better lateral root development and higher level of accumulation of the isoform OsTIP2;1 under prolonged osmotic stress. This accumulation was detected in almost all root cells at lateral root initiation zone and abundantly in the endodermis of radicle and lateral roots at mature zone (Matsunami et al., 2016). Moreover, osmotic stress markedly enhanced the OsPIP protein abundance in the roots of both lowland (Xiushui 63) and upland (Zhonghan 3) rice cultivars and in leaves of upland cultivar (Lian et al., 2006). However, a remarkable reduction of osmotic Lpr of two rice cultivars upon both drought and salt stress was reported (Meng and Fricke, 2017).

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Redistribution of PM aquaporins upon salt and drought stress

This set of markers was also used for a dynamic survey of membrane compartments upon environmental challenges. To investigate the behaviours of aquaporins upon salt and osmotic stress, roots of transgenic lines expressing either OsPIP1;1, OsPIP2;4 or OsPIP2;5 constructs were challenged with 100 mM NaCl or 20% (w/v) PEG6000 for 30 min and observed by confocal microscopy. Firstly, we validated the behaviour of the OsPIP constructs in Arabidopsis and observed a specific relocalization of these PM aquaporins into intracellular compartments depending on the isoform, the cell type or the treatment (Figure S3). For instance, OsPIP2 isoforms exhibited a stronger tendency to relocalize upon abiotic stress than OsPIP1;1. Secondly, when expressed in rice crown root cells, the OsPIP constructs exhibited a marked intracellular labelling (Figure 3). In any case, both the NaCl and PEG stresses resulted in a marked increase of intracellular labelling as compared to control conditions. For instance, in exodermal cells, we noticed that OsPIP1;1 construct labelled a compartment surrounding the nucleus, tentatively identified as the endoplasmic reticulum. Redistributions of OsPIP2;4 and OsPIP2;5 were observed mainly in punctuated structures. In Arabidopsis, upon drought stress, a RING membrane-anchor E3 ubiquitin ligase has been reported to be involved in the ubiquitination of AtPIP2;1 and the retention in the ER of this aquaporin (Lee et al., 2009). In mesodermal cells, intracellular labelling with the OsPIP1;1 construct was punctuated and detected in only 2% cells in control condition but in ~55% and 43% of cells, under salt and osmotic stress, respectively. The differences of subcellular redistribution of OsPIP1;1 and OsPIP2s; upon stress; between exodermal and mesodermal cells suggested a isoform-specific and cell-specific response. We observed a similar phenotype in mesodermal cells of radicle root (Figure S4). The CFP-LTi6a construct showed a much lower tendency to relocalize in intracellular compartments upon salt or osmotic stress (Figure S5). Importantly, a specificity of OsPIP relocalization upon these stresses was observed in rice, whereas such phenomenon was not reported for AtPIP in Arabidopsis (Boursiac et al., 2008). Following the description of AtPIP internalization upon salt and oxidative stress in Arabidopsis roots (Boursiac et al., 2008), the present work extends this behaviour to their orthologues in rice. Since this phenotype can be observed in two representative dicot and monocot species, we propose that it represents a conserved adaptive mechanism upon abiotic environmental stress.

Molecular cloning of membrane protein markers and plant transformation

Molecular cloning information is summarized in supplementary Table S1. OsPIP1;1 was subcloned into pDONR207 and transferred into the destination vector pGWB5 (Nakagawa et al., 2007) using Gateway® Gene Cloning technology (Invitrogen, USA), according to the manufacturer’s instruction. The whole set of the other protein markers were subcloned into pBluescript SK vector (Stratagene, USA) or pUC57 (Table S1), and then cloned into the pGreenII 0179 binary vector (Hellens et al., 2000) under the transcriptional and translational control of a double enhanced cauliflower mosaic virus 35S promoter and the 3’ end of the pea ribulose-1,5-bisphosphate carboxylase small subunit rbcS gene. A molecular construct consisting of the maize ubiquitin-1 promoter controlling the expression of a fluorescent plasma-membrane-localized fusion protein (ECFP-LTI6a) (Cutler et al., 2000) was obtained by synthesis (Genscript) and cloned into the plasmid pUC57. The insert was released by a double digestion with EcoRI et KpnI and cloned into the binary vector pCAMBIA2300 linearized by EcoRI and KpnI. All constructs were confirmed by DNA sequencing by Eurofins Genomics (Germany). The recombinant DNA plasmids were electroporated into Agrobacterium tumefaciens strain EHA105 or GV3101 for rice or Arabidopsis transformation, respectively. Japonica rice Nipponbare cultivar was transformed according to a modified seed-embryo callus transformation procedure (Sallaud et al., 2003). Arabidopsis transformation was performed according to flower dip protocol (Clough and Bent, 1998). Selection of transgenic plants was performed with medium supplemented with hygromycin.

Table of contents :

General Introduction
1. Drought and salt stress in plant with a central focus on rice
2. Rice root architecture and anatomy
2.1 Rice root architecture
2.2 Rice root anatomy
3. Diversity of AQP isoforms and their substrate specificities in rice
4. Rice AQP expression in normal conditions
4.1 Tissue-specific expression
4.2 Diurnal-specific expression
5. Role of AQPs in the response of rice to drought and salt stress
5.1 Inhibition of root hydraulic properties upon drought and salt stress
5.2 Regulation of AQP expression in rice upon drought and salt stress
5.3 Genetic manipulation of AQPs in rice
6. Context and thesis objectives
References
Chapter I: Sub-cellular markers highlight intracellular dynamics of membrane proteins in response to abiotic treatments in rice
Abstract
Introduction
Results and discussion
Rice transgenic line creation and subcellular localization visualization
Redistribution of PM aquaporins upon salt and drought stress
Dynamic of endocytosis of PM aquaporins upon salt stress
Materials and methods
Molecular cloning of membrane protein markers and plant transformation
Plant materials and growth conditions
Confocal microscopy visualization
ClearSee tissue preparation
Stress application and pharmacological approach
Declarations
Supplementary figures
Supplementary table
References
Chapter II: Genetic manipulations of rice to improve drought and salt tolerance.
Results
Characteristics of transgenic plants compared to wild-type in control condition
Characteristics of transgenic plants compared to wild-type upon drought stress
Characteristics of transgenic plants compared to wild-type upon salt stress
Discussion
Materials and methods
Experimental design
Fraction of transpirable soil water
Leaf water content
Chlorophyll content
Leaf water potential
Stomatal conductance
DAB staining
Na+ and K+ content measurements
Statistical analysis
Acknowledgments
References
Chapter III: Control of water uptake by root system architecture in rice (Oryza sativa L.) 
Abstract
Introduction
Results
Root morphological characteristics
Root hydraulic conductance and conductivity
Effects of azide treatment on Lpr
Effects of China ink treatment on Lpr
Effects of salt stress on the root hydraulics
Covariation of root hydraulic properties and root morphology
Discussion
Root morphological characteristics
Root hydraulic properties
Effects of salt stress on the root hydraulics
Towards a global understanding of root hydraulics strategies in rice
Conclusion
Materials and methods
Plant materials and growth conditions
Root phenotyping
Root hydraulic property measurements
Statistical analysis
Acknowledgments
References

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