The Impacts of TRR14-Overexpression on Arabidopsis thaliana Growth and Photosynthetic Parameters

Background TRR14 protein is a small protein, a member of a multigene family in Arabidopsis which was found as the fi rst protein during screening seedlings for their resistant to the trehalose sugar. Objectives A number of TRR14-overexpressing plants were subjected to the characterization in the present research, among which, the associated morphological features and changes accompany growth pattern and photosynthesis related parameters. Materials and Methods TRR14 gene was isolated from Arabidopsis Thaliana and cloned into the pBin-35S vector. Recombinant vector was transferred to the Arabidopsis (Col-0) via Agrobacterium tumefaciens using the Floral Dipping method. Seeds from the TRR14 overexpressed (TRR14) and the Col-0 wild-type (WT) plants were shown on soil under long day conditions. Several measurements were then performed including determination of the fresh and dry weights, leaf area, chlorophyll a and b (Chl a and Chl b) content, Chl a/b ratio, total chlorophyll and carotenoids content, soluble and insoluble sugars content, total and soluble protein content, the Hill reaction rate, chlorophyll fl uorescence, as well as photorespiration rate. Meanwhile, the chloroplastic proteins were investigated by SDS-PAGE analysis. Results TRR14 plants showed a signifi cant increase in fresh and dry weights, leaf area, and total and soluble protein content along with a signifi cant decrease in the insoluble sugar contents was observed in comparison to the WT plants. Chl a, Chl b, total chlorophyll content, Chl a/b ratio, carotenoids content, Hill reaction rate, and chlorophyll fl uorescence didn’t show a signifi cant diff erence between TRR14 and WT plants. The SDS-PAGE gel electrophoresis of the chloroplastic proteins showed a thick band with a molecular mass of 25 kDa in TRR14-overexpressed plants, compared to the WT plants. Remarkably, photorespiration rate was decreased in TRR14 plants compared to WT plants. Conclusion The increased biomass of TRR14 transformed plants might be due to its ability in reducing photorespiration through concentrating CO2 in the leaf’s intercellular spaces.


Background
Encoded as At4g10300, TRR14 (Trehalose Resistance) protein is 139 aa in length and contains Cupin domain (1,2). In plants, proteins containing conserved Cupin domain play diverse functions in the metabolism and signaling. Enzymes such as: phosphomanoseisomerase, polyketide synthase, dioxygenase, oxalate oxidase (germins), and auxin binding protein belong to this group (2,3). So far 18 diff erent functional Cupin subclasses were identifi ed in plants (2). TRR14 has six paralogues in Arabidopsis and even more orthologous in Oryza sativa, and Medicago sativa. Beyond plants, the closest TRR14's homologs are found in the bacteria, particularly in many cyanobacteria including Synechococcus sp. and Prochlorococcus marinus. This protein has been localized in the chloroplast and its expression is ubiquitous (4).
Trehalose metabolism has recently been recognized to play an important role in carbon signaling in the plants (5,6,7). Trehalose is the alpha, alpha-1-1 linked glucose disaccharide, which is found ubiquitously and is being thought to be evolutionary ancient (8).
The synthesis of the trehalose-6-phosphate (T6P), the immediate precursor of the trehalose, is indispensable for development and carbon utilization in Arabidopsis seedlings (9). Meanwhile, T6P is a signal for sucrose status and regulate the content of sucrose in the plant (10,11). Plants overexpressing trehalose-6-phosphate synthetase (TPS) display an increased T6P and typically have dark green cotyledons, leaves, as well as an increased photosynthetic capacity per leaf area unit (12).
Photorespiration is an indispensable cleavage of ribulose 1, 5-bisphosphate (RuBP) by the molecular oxygen which leads to an equimolar production of 3-phosphoglycerate (3-PGA) and 2-phosphoglycolate . This process occurs because Rubisco (i.e. the enzyme which catalyzes the carboxylation of RuBP) is also able to oxygenate this compound. While 3-PGA can easily be utilized by the Calvin cycle for the production of sugars, the two-carbon compound 2-PG is toxic and its removal from photosynthesizing cells requires a long metabolic pathway known as photorespiratory carbon cycle which is accompanied by the release of CO 2 (13). The Photorespiration-mediated CO 2 release imposes yield loss on most of the highly bred crops plants, particularly when experiencing heat or drought stresses which favor CO 2 shortage and O 2 accumulations in photosynthesizing mesophyll cells due to stomata closure (14,15).

Objectives
TRR14 is a novel protein and its function is not precisely understood. Our previous results showed that TRR14 might be involved in tolerance of the plants toward salinity and drought stresses (4,17), as plants with the capacity to restrict their photorespiration are more effi cient in tolerating these two type of stresses (17,18,19,20). In the present research, the impact of TRR14 overexpression was evaluated on Arabidopsis thaliana with respect to several morphological and physiological parameters related to the photosynthesis and photorespiration. The results obtained from such studies may lead to a better understanding of the possible links between trehalose metabolism, photosynthesis, and photorespiration.

Wild-type Plants and Transgenic Lines' Growth
Seeds from Arabidopsis thaliana wild-type (WT) plants ecotype Columbia-0 (COL-0) and transgenic lines (TRR14) were planted in compostsoil (Takidar-Iran). Plants were grown in the controlled growth chamber with a light normal irradiance of 150 μmol photon m -2 s -1 in a light-dark cycle of 16/8 hours at 25°C.

RNA Extraction and cDNA Synthesis
Plant material was immediately frozen in the liquid nitrogen and was ground to the powder in a dismembrator (Braun, Melsungen, Germany). The total RNA was obtained using the RNeasy plant mini kit, according to the instruction of the manufacturer (QIAGEN USA, Valencia, CA). The quantity and quality of the extracted RNA were determined using spectrophotometeric method and agarose gel electrophoresis, respectively. Genomic DNA contamination was removed with DNAse I (DNA-free, Ambion, Austin, USA) treatment. The absence of DNA was analyzed by performing a PCR reaction (40 cycles) on the DNase I-treated RNA, cDNA synthesis, and PCR amplication using Taq DNA polymerase and specifi c primers as follows. cDNA synthesis was performed using M-MLV reverse transcriptase (Promega, Madison, WI) on 1ng of the total extracted RNA with 0.5 μg of oligodT16v (custom oligo from Invitrogen, Carlsbad, CA) and 0.5 μg of random hexamer (Invitrogen, USA). PCR was performed with forward and reverse primers (TRR14F: 5 ' -ACCCAACTCGGTGTTCGTAG-3 ' and TRR14R: 5 ' -TGATAGCAGCCATTCACTAG-3 ' ).

Plasmid Construction
The obtained PCR fragments were cloned into the pGEM-T Easy vector (Promega, Madison, USA) and sequenced. The fragment was then isolated and purifi ed from the pGEM-T easy vector clones and cloned into the pBin19 expression vector using standard restriction/ ligation techniques (HindIII/EcoRI). The CaMV 35S promoter was isolated by digestion with EcoRV from the pUC-18 vector and subsequently ligated into pBin19 (HindIII/EcoRI).

Plant Transformation
The construct was transformed into Agrobacterium Tumefaciens Gv3101 by using a Gene Pulster electroporator (BioRad, Hercules, Ca, USA), according to the manual. Then, Arabidopsis thaliana ecotype Col-0 plants were transformed by the fl oral dip method (21). Transgenic T1 seedlings were selected on half MS medium supplemented with the 50 mg.L -1 Kanamycin. A transformation with the full-length cDNA of TRR14 has produced 20 independent lines.

Quantitative Real Time PCR Analysis
Total RNA from ov rexpressed and the WT plants were isolated using RNeasy plant mini kit (QIAGEN USA, Valencia, CA). After cDNA synthesis, Quantitative-PCR (Q-PCR) was performed using ABI-prism 7700 Rezapoor H. et al.
Sequence Detection System (PE-Applied Biosystems, Foster City, CA) according to the standard SYBR green PCR Master Mix (Applied Biosystems, UK) and specifi c primers (TRR14F: 5 ' -AACTTGATCGGGATATGGAGTG-3 ' TRR14R: 5 ' -AACTTGATCGGGATATGGAGTG-3 ' ). The Actin gene(At3g18780)was used to serve as house the keeping gene and the relevant primers for measuring its expression level were as following sequences: AtACTIN2F: 5 ' -GACCCAAAGACGGAGACTCTT-3 ' and AtACTIN2R: 5 ' -GCCAAGTGATTGTGGAGACTC-3 ' . Each experiment was repeated 3 times. Gene expression was normalized to the expression of AtACTIN2as the calibrator reference gene, using the comparative Ct method (22). Melting curve analysis was done for checking the specifi city of the PCR reaction and lack of primer dimers, as well. For further confi rmation, PCR products were loaded onto a %2 agarose gel, ran, and visualized.

Pigment Content and Fluorescence Measurements
Chlorophylls a, b, total chlorophyll, and carotenoids' contents were determined essentially as described by Jeff ery and Humphrey (23). Leaves from the 3 weeks old plants were frozen in liquid nitrogen and ground in 80% (v/v) acetone. The absorbance was then measured at 670, 645, and 663 nm (Shimadzu UV-160). The chlorophylls a, b, total chlorophyll, and carotenoids' content were then calculated according to the following formula: The fl uorescence of Chlorophyll a was determined with OPTI-Sciences OS-30 fl uorometer (Walz, Eff eltrich, Germany). After 15 min adaptation of the Arabidopsis plants to the dark, F 0 (the initial fl uorescence content of PSII reaction center) was determined in the presence of 10 μmol photons m.s -1 measuring beam. The Fm (maximum fl uorescence content in the darkadapted state) was determined using a 0.8 s saturating irradiance pulse. The fl uorescence parameter Fv/Fm was calculated using the DualPAM software.

Chloroplast Preparations and Measurement of the Hill Reaction Rate
The rate of Hill reaction in the chloroplast preparations was determined according to the Trebst (24). Chloroplasts were extracted from the frozen ground leaf material in the ice-cold buff er consisting of 20 mM Tris-HCl (pH 7.5), 0.3 M sucrose, 10 mM EDTA and 5 mM MgCl 2 . The resulting crude extract was fi ltered and then centrifuged at 3500 ×g for 5 min. The obtained pellet was re-suspended in 10 mLof the ice-cold homogenate buff er thoroughly with a Pasteur pipet. The chloroplast suspension was stored on ice all the times.
The Hill reaction rate in the prepared chloroplast was determined spectrophotometrically by measuring absorbance at 600 nm due to the reduction of Dichlorophenolindophenol (DCPIP). The Hill reaction rate was expressed as the changes in absorbance per milligram chlorophyll per minute.

Carbohydrate and Protein Measurement
The soluble and insoluble sugars' content was determined spectrophotometrically, as described by Kochert (25). The soluble and the total proteins of the leaf were determined according to the methods to the Bradford (26) and Markwell (27), respectively.

SDS-PAGE Analysis of the Chloroplast Proteins
The SDS-PAGE of the chloroplast protein samples was carried out according to Flingand Gregerson (28). The chloroplast preparation was suspended in the 100 μL of the homogenization buff er. After adding 5.0 mL n-Hexan:2-propanol (3/2; v/v), it was centrifuged at 4000 ×g for 15 min. Then the precipitated protein was washed with 5.0 mL acetone (80% v/v) and dried under a stream of the nitrogen gas. The dried protein precipitates were fractioned by SDS-PAGE using 15% w/v polyacrylamide gels that were subsequently stained with the Coomassie blue (R250, Sigma, Germany).

Photorespiration Measurement
Plants were grown in soil under a short day regime at 22 o C. The setup was used to test the diff erences between the response of TRR14 overexpressed and WT plants to the air with 2% oxygen content and room air with the normal 21% oxygen content. The ratios of the leaf internal versus air CO 2 concentrations were measured in the WT and in the overexpressed TRR14 plants. Photorespiration was calculated by using the following formula: (Vo+Rp)/(Vo+Vc), where Rp is the photorespiratory CO 2 release, Vc the rate of carboxylation and Vo the rate of oxygenation (29).

Statistical Analyses
The data were analyzed by statistical SAS package (version 9). The reported values were means of the three replicates. The Duncan's test was used to compare means for the signifi cance.

Overexpression of TRR14
To investigate the physiologic role of TRR14, the corresponding cDNA was integrated into a binary vector pBin19 under the control of CaMV 35S promoter and then transformed into the WT (Colombia-0 ecotype) seedlings. The full-length cDNA transformation into Arabidopsis Col-0 plants provided 20 independent lines which showed resistance to the selectable marker. Three independent transgenic lines were selected for TRR14 gene expression analysis (T7, T12, and T17). There were not non-specifi c amplifi cation and the signs of primer dimers in the PCR reactions (Figs. 1-A, B), so, gene expression analysis was valid. The transcript content of TRR14 was more than 2 times higher in the transformed lines compared to the WT ( Fig. 1-C). We used T17 transgenic line for further analysis.

Characterization of TRR14 Overexpressed Versus WT Plants.
The TRR14 overexpressed and WT plants showed diff erences in the morphology, leaf area, fresh, and dry weight when grown in soil. While there was no signifi cant diff erence in the height between TRR14 overexpressed and the WT plants ( Fig. 2A), the former showed a higher leaf area compared to the WT (Fig.   2B). Furthermore, the TRR14 overexpressed plants were not diff erent with respect to the fl owering time when compared with the WT ones. The TRR14 overexpressed plants had signifi cantly greater fresh and dry weights than that of the WT plants. The fresh and dry weights were 0.19 ± 0. 1 and 1.4 ± 0.01 g respectively in the TRR1overexpressed plants, while they were 0.8 ± 0.005 and 0.8 ± 0.09 g in the WT plants ( Table 1).
The Chl a, Chl b, total chlorophyll, carotenoid content, and Chla/b ratio were similar in both TRR14overexpressed and WT plants, as well ( Table 1). The effi ciency of the PSII (i.e. Fv/Fm value and the Hill reaction rate which represents the plant water oxidation capacity) was almost indiff erent between TRR14 overexpressed and the WT plants (Fig. 3B). Analysis of the carbohydrate showed that soluble sugar content was similar in both TRR14 and WT plants (Fig. 4A). But, insoluble sugar content was signifi cantly greater in the WT than that of the TRR14-overexpressed plants (Fig.  4B).  Rezapoor H. et al.

The Chloroplast Protein Composition of the TRR14-overexpressed and the WT Plants
The leaf materials from the TRR14-overexpressed and WT plants were analyzed for the soluble and total protein content in addition to chloroplast protein composition. There were signifi cant diff erences in the soluble and total protein content between TRR14overexpressed and WT plants. The soluble and total protein contents were lower in the WT compared to theTRR14-overexpressed plants (Figs. 4C, 4D).
Then, isolated proteins from chloroplasts of TRR14 overexpressed and WT plants were analyzed through SDS-PAGE. The protein band patterns of the both TRR14 overexpressed and WT chloroplasts were similar. However, SDS-PAGE fractions detected one extra protein band corresponding to the 25 kDa in the transgenic line (Fig. 5).

Photorespiration is Altered by Overexpression of TRR14
To investigate whether the TRR14 overexpression has any eff ect on the plant photorespiration, we determined the CO 2 concentrations ratios of the leaf internal spaces versus air in both WT and in the overexpressed TRR14 plants. Measurements of the ratio of the leaf internal CO 2 (Ci) and ambient CO 2 (Ca) concentration Table 1. The eff ect of TRR14-overexpression on several growth related features and photosynthetic pigments content. Fresh weight (FW), dry weight (DW), Chlorophyll content (Chl), Chlorophyll ratio (Chl a/b), and carotenoid content of the Arabidopsis wild-type (WT) and TRR14-overexpressed (TRR14) plants that were grown under a normal condition.   (Ci/Ca) revealed that the gas exchange process of TRR14 overexpressed plants is diff erent from that of WT ones (Fig. 6A). The results further revealed that photorespiration is signifi cantly reduced in the TRR14overexpressed plants (Fig. 6B).

Discussion
The metabolism of trehalose is indispensable in the plants (30). Trehalose related metabolites are present in only very low concentrations and their role in plants is not fully understood so far (5). A minor alteration in the steady state content of T6P; the precursor of trehalose in the plants was shown to result in dramatic and pleiotropic phenotypic changes of the plants (12,31). Trehalose supplied to the growth medium of the seedlings inhibits their growth and alteration in the carbon allocation in the seedlings so that starch is preferentially accumulated in the shoots rather than the roots (32, 1). However, TRR14 overexpression provides resistance to the both growth inhibition and starch accumulation eff ects of T6P (1). The results obtained here have revealed that TRR14-overexpressed plants display a higher leaf area, fresh, and dry weight as compared to the WT plants. These results suggest that TRR14 may enhance the growth of Arabidopsis.
Although the starch content of TRR14-overexpressed plants was lower than WT, they had greater amounts of both soluble and total proteins which may indicate an alteration in C/N metabolism of the overexpressed plants in favor of organic nitrogen production. T6P is known to aff ect photosynthetic capacity per unit leaf area (12). Accordingly, photosynthesis might be a target of T6P functioning. Neither chlorophylls and carotenoids contents nor the rate of Hill reaction and photosynthetic effi ciency (Fv/Fm) was appeared to be diff erent between TRR14-overexpressed and WT plants (Fig. 3). These suggest that the positive eff ects of TRR14 overexpression on plant growth is not related to its possible eff ects on light energy absorption and/ or transduction through chloroplast pigment complexes and electron transport chain. The current result showed that TRR14 overexpressed plants have a higher Ci with associated lower RP. This data revealed that overexpression of TRR14 leads to the reduced rate of photorespiration in the plants (Fig. 6).
Naturally, some plants which display C 4 , C 3 -C 4 intermediate, and CAM metabolisms down-regulate their photorespiration through concentrating CO 2 at the site of carboxylation where Rubisco resides (i.e. chloroplasts, and at a larger scale the leaf's intercellular spaces) (33). Measurement of the CO 2 concentration in the leaf internal spaces of the TRR14 overexpressed plants has justifi ed their reduced rate of photorespiration. The transformed plants had signifi cantly greater contents of the internal CO 2 concentration than the WT plants (Fig. 6), thus, TRR14 functioning in reducing photorespiration is somehow accompanied with or through an increased CO 2 concentration in the leaf intercellular spaces. This might explain the reason for observing a greater biomass of the transformed plants.  The mechanism through which TRR14 brings about an increased concentration of intercellular CO 2 needs further investigation. This protein might aff ect leaf cell development and/or metabolic compartments in favor of localized CO 2 production.
Such a condition has been reported in Panicum milioides and Moricandia arvensis, the two C 3 -C 4 intermediate species which their proximal chlorenchymatous cells contain high activity of glycine decarboxylase (i.e., a major CO 2 releasing enzyme of the photorespiratory pathway) while their distal cells have a high Rubisco activity which benefi t from the released CO 2 by the proximal cells (34,35). It has been suggested that photorespiration imposes yield ceilings on most of the high-bred crops planted (33,35). However, in stress conditions such as excess light, drought, or possibly other stresses that aff ect guard cell functions, this process has some advantages for the C 3 plants by ensuring their survival rather than productivity (36). Despite cloning of Arabidopsis genes which encode photorespiration pathway basic enzymes, questions regarding the regulation of photorespiration are still open and demand further scrutiny (37). Attempts to reduce photorespiration have mostly failed even though it is a major goal for crop improvement. An increased understanding of the photorespiration regulation is needed in the context of the climate change as stress-induced by drought and heat gradually increase photorespiration of the existing crops and lead to yield loss (38).
So far, the physiological function of TRR14 has largely been unknown, except the fi nding that TRR14 overexpression leads to the salt and drought stress resistant (4,16). Our previous data has indicated that TRR14 overexpressed plants had unchanged seed germination, root length, and chlorophyll content under the salt and drought stresses. In addition, the activity of several enzymes related to the scavenging reactive oxygen species like peroxidase and catalase were signifi cantly induced in TRR14 overexpressed under salt and drought treatment (4). The reduced photorespiration, as observed here in the TRR14 transformed plants, implies also the reduced H 2 O 2 production during photosynthesis which is in favor of a greater biomass production (1,4). This feature reduces plant's energy consumption for removal of various reactive oxygen species. Further studies are needed to reveal the roles played by TRR14 in reducing plant photorespiration and tolerance to the environmental stresses. The obtained data could un-doubtfully be useful in the near future for the production of crops with greater yield performance.
It has been shown that photorespiration has an important role in the protection of photosynthetic apparatus against stress (39,40). This possibility needs further exploration since T6P and trehalose have been shown to convey stress-resistance responses that could be mediated by specifi c reactive oxygen signaling (41)(42)(43)(44).

Conclusions
In conclusion, TRR14 is a novel protein important for the regulation of photorespiration, an area of research of great economic and environmental interest. With global climate change and an increasing agricultural production on marginal lands, the link between TRR14, trehalose metabolism, and photorespiration regulation is of interest for future studies.