A Transaldolase From Aquilaria Sinensis Involves in Aba-mediated Seed Germination and Root Growth

Transaldolase, the key enzyme of the pentose-phosphate pathway, plays an important role in plant growth and defense. Seed germination is a key factor that inuences the cultivation of Aquilaria sinensis, the plant source of agarwood, which is widely used as a traditional medicine, perfume and incenses. However, little is known about the function of transaldolase in abscisic acid (ABA)-mediated seed germination. In the present study, the full-length AsTal1 gene was isolated and characterized from A. sinensis calli. Sublocalization analysis indicated that AsTal1 was localized in the cytoplasm. In addition, phenotypic analysis indicated that AsTal1-overexpressing Nicotiana benthamiana (OE) plants were less sensitive to ABA during seed germination and root growth than wild-type (WT) plants. Overexpression of AsTal1 regulated the expression of genes involved in ABA metabolism, biosynthesis and signal transduction under ABA treatment. In addition, expression of NbRbohA and NbRbohB was inhibited in the overexpression lines, whereas the abundance and activities of the antioxidative enzymes SOD, APX, and POD were higher in the transgenic plants than in the WT lines after ABA treatment. Taken together, our results indicated that AsTal1 is involved in the ABA response during seed germination and root growth by regulating the expression of genes involved in the ABA signaling pathway and the enzymes responsive to ROS. we the of under a vital root growth in Arabidopsis results suggested that AsTal1 overexpression could enhance antioxidant enzyme activities and increase expression levels to remove excess ROS under ABA treatment.


Introduction
The oxidative pentose-phosphate pathway (PPP) plays an essential role in carbohydrate metabolism, by both serving as a source of NADPH for biosynthesis and balance of reactive oxygen intermediates in plant cells and providing precursors for several main biosynthesis pathways (Hawkins et al. 2018;Yang et al. 2015). The OPPP comprises two separate branches: an oxidative branch, in which glucose-6phosphate dehydrogenase (G6PDH) and 6-phosphogluconate dehydrogenase generate ribulose 5phosphate (Ru-5-P) and NADPH, and a nonoxidative branch, in which transketolase (TK) and transaldolase (TAL) convert Ru-5-P into xylulose 5-phosphate (Xul-5-P) and ribose-5-phosphate (Rib-5-P) (Caillau and Paul Quick 2005;Hawkins et al. 2018;Stincone et al. 2015). This suggests that the main regulatory steps of the OPPP are catalyzed by TAL (Caillau and Paul Quick 2005). TAL is ubiquitous in prokaryotes and eukaryotes and was rst identi ed as the rate-limiting enzyme in yeast (Yang et al. 2015). In humans, TAL de ciency causes pathological disorders such as hydrops fetalis, liver dysfunction, and neonatal multiorgan disease (Michel et al. 2015;Perl et al. 2011). In plants, TAL appears to be involved in plant defenses and development. For instance, TAL abundance increased in both cucumber leaves and wheat leaves upon infection by fungi (Caillau and Paul Quick 2005). The expression level of TAL in potato also increased in response to stress (Moehs et al. 1996). Yang et al.
recently indicated that knockout of TAL in rice resulted in a dwarf phenotype, with narrow, short leaves and an altered vascular pattern (Yang et al. 2015). Zheng et al. reported that GSM2, a TAL from Arabidopsis thaliana, is involved in maintaining reactive oxygen species balance in response to Glc during seeding growth (Zheng et al. 2020).
ABA is a key phytohormone that participates in the response to environmental stresses and plant growth, such as seed germination and dormancy and root growth and development (Du et al. 2019;Fujita et al. 2011; Liu et al. 2015). The promoters of ABA-mediated genes contain conserved ABA-responsive elements (ABREs) with the core nucleotide sequence ACGT, which is the binding site for basic leucine zipper (bZIP) transcription factors (Hattori et al. 2002). In Arabidopsis, the transcription factors ABI3 and ABI5 play important roles in mediating the ABA signaling pathway during seed germination and dormancy (Hattori et al. 2002;Kashiwakura et al. 2016 Table 1). The resulting DNA fragments were ligated into a pMD19-T vector, and three clones were sequenced in both directions. The ORFs of the cDNA sequence of AsTAL1 were predicted with the ORF nder online tool (http://www.ncbi.nlm.nih.gov/projects/gorf/). The amino acid alignments were performed by DNAMAN software, and phylogenetic trees were constructed using the neighbor-joining tree algorithm of the MEGA 6.0 program.

Subcellular localization
The ORF of AsTal1 was isolated by PCR ampli cation using speci c primers (Table S1). The fragments were fused to the C-terminus of the green uorescent protein (GFP) gene in a pCAMBIA1300-35S-EGFP binary vector. The binary vectors were then transformed into Agrobacterium tumefaciens strain EHA105, after which the transformed Agrobacterium were in ltrated into Nicotiana benthamiana leaves as described in previous reports. Subcellular localization was observed via laser confocal scanning microscopy.

Quantitative real-time PCR (qRT-PCR) assays
qRT-PCR was performed on a CFX Connect TM Real-time System (Bio-Rad) using TransStart Tip Green qPCR SuperMix (Transgen) according to the manufacturer's protocol. The A. sinensisGAPDH gene and the N. benthamiana β-actin gene were used as internal controls to normalize RNA levels. Real-time PCR was initiated with 30 s of incubation at 94°C, followed by 40 cycles of 94°C for 5 s and 60°C for 30 s. The primers used for qRT-PCR were generated by the Primer 5.0 program (Table S1), and the 2 -ΔΔCT method was used to analyze the expression level of AsTal1 genes. Three biological experiments and three experimental replicates were included to analyze gene expression. The primers used for qRT-PCR are listed in Table S1.

N. benthamiana transformation
To construct AsTal1 overexpression vectors, the AsTal1 ORF was obtained by speci c primers and subsequently subcloned into a pCAMBIA1300-35S binary vector to yield pCAMBIA1300-35S-AsTal1 vectors. The pCAMBIA1300-35S-AsTal1 plasmids were then transformed into wild-type N. benthamiana using the Agrobacterium-mediated leaf disc transformation method. Positive transgenic N. benthamiana plants were identi ed through PCR and qPCR. T3 seeds of three independent overexpression lines (OE) and wild type (WT) plants were used to analyze the function of the AsTal1 gene.

Subcellular localization of AsTal1
To determine the subcellular localization of AsTal1, the coding sequence of AsTal1 was cloned into a pCAMBIA1300-35S-EGFP vector. The recombinant vectors pCAMBIA1300-35S-EGFP-AsTal1 and pCAMBIA1300-35S-EGFP were subsequently transformed into N. benthamiana epidermal cells by the agroin ltration method. After two or three days of incubation, the expression of the introduced gene was examined via laser confocal scanning microscopy. Confocal microscopy of the N. benthamiana epidermal cells of the in ltrated leaves indicated that GFP:AsTal1 was localized in the cytoplasm ( Fig. 2A), whereas the control GFP was distributed across the whole epidermal cell (Fig. 2B).

Expression pro les of AsTal1 in different tissues and in response to abiotic stresses
To determine the expression of AsTal1 in different tissues, relative quantitative real-time PCR analysis was performed on the total RNA from the roots, stems, leaves and shoot tips (Fig. 3A). The results showed that AsTal1 was expressed in all of the tested tissues, with tissue-speci c expression patterns. AsTal1 expression was shown to be highest in the shoot tips, followed by the roots and stems. To investigate the putative role of AsTal1, the transcript levels after exposure to ABA, salt, drought, cold temperature and heavy metal stress were measured. As shown in Fig. 3B Fig. 4A-4B, the germination rates of the OE and WT lines were similar under control conditions. Seed germination was signi cantly inhibited for both OE and WT lines in the presence of 1 and 2 µM ABA, but the suppression of OE plant germination was much weaker than that of the WT. For instance, the germination of OE lines reached 85.5% compared with 64.0% for WT line after 5 days of treatment with 1 µM ABA, while 62.6% of the OE seeds compared with 36.7% of the WT seeds after 5 days of treatment with 2 µM ABA (Fig. 4B). To further con rm the transgenic phenotypes, the primary root length was measured. Under normal growth conditions, no signi cant difference in primary root growth was detected between the WT and three OE lines (Fig. 4C-4D). However, the primary root growth of the OE plants was longer than that of the WT lines after treatment with 10 µM ABA (Fig. 4D), indicating that the OE lines were hyposensitive to ABA treatment.

AsTal1 affects genes involved in the ABA signaling pathway
To determine the function of AsTal1 in the ABA signaling pathway, we examined the expression of ABA signaling-related genes, including those involved in ABA biosynthesis, catabolism and signal transduction. The qRT-PCR results showed that the expression level of genes involved in ABA biosynthesis (NCED) was lower in the OE lines than in the WT plants under ABA treatment (Fig. 5A). In contrast, the catabolism-related genes CYP707A1 and CYP707A2 presented higher expression levels in the OE lines than in the WT under ABA treatment (Fig. 5B-5C). Furthermore, our results indicated that the transcript levels of the ABA signaling-related transcription factors ABI3 and ABI5 signi cantly decreased in the OE lines after ABA treatment (Fig. 5D-5E). These results suggested that AsTal1 is involved in the regulation of ABA metabolism and signal transduction.

Overexpression of AsTal1 reduces ROS levels under ABA treatment
A previous investigation showed that ABA causes ROS production and oxidative damage. To further determine the effects of AsTal1 on ABA responses, ROS accumulation was evaluated. Our results indicated that the H 2 O 2 content in the OE lines was less than that in WT lines and that the O 2 − content signi cantly decreased in the OE lines ( Fig. 6A-6B). In plants, ROS can be generated by amino oxidases, oxygen photoreduction and peroxidases. In plants, the main ROS are produced by NADPH oxidases, which play an important role in primary root growth under different stresses. To determine whether the function of AsTal1 in response to ABA is involved in the NADPH oxidase pathway, we analyzed the expression of NADPH oxidase genes in OE and WT lines treated with ABA. As shown in Fig. 6C-6D, the expression levels of NbRbohA and NbRbohB signi cantly increased after ABA treatment in the WT and OE lines. However, the NbRbohA and NbRbohB expression levels in the OE lines were markedly lower than those in the WT line under ABA treatment. These results suggested that AsTal1 is involved in NADPH oxidase-dependent ROS production; antioxidant enzymes remove extra ROS to maintain the balance between ROS production and scavenging under different environmental stresses. Furthermore, we measured the expression and activity of antioxidant enzymes in OE and WT lines with or without ABA treatment. The results indicated that ABA-induced transcript levels and activity of SOD, APX and POD in the OE lines were signi cantly higher than those in the WT plants ( Fig. 6E-6I). These results indicate that AsTal1 could enhance the capacity to scavenge excess ROS by regulating the expression and activity of antioxidant enzymes.

Discussion
A. sinensis is the main plant species that produced agarwood used as traditional medicine, incense and perfume (Ding et al. 2020 induced by abiotic stresses such as salt, drought, cold and heavy metal stress. Furthermore, the expression level of AsTal1 was signi cantly induced by ABA treatment, suggesting that AsTal1 functions in ABA signaling. ABA is an important phytohormone that regulates seed dormancy and germination and root development (Chen et   Multiple sequence alignments of TAL sequences were performed using ClustalX, and the phylogenetic tree was constructed using MEGA6 with the neigbor-joining (NJ) method and 1000 bootstrap. The TAL proteins were classi ed into two groups (I, II).