Chloroplast thylakoid ascorbate peroxidase PtotAPX plays a key role in chloroplast development by decreasing hydrogen peroxide in Populus tomentosa

Abstract

Chloroplast development is a complex process that is critical to the growth and development of plants. However, the detailed mechanism of chloroplast development in woody plants remains unclear. In this study, we showed that chloroplasts with elaborate thylakoids could develop from proplastids in the cells of calli derived from leaf tissues of Populus tomentosa upon exposure to light. Chloroplast development was confirmed at the molecular and cellular levels. Transcriptome analysis revealed that genes related to photoreceptors and photosynthesis were significantly up-regulated during chloroplast development in a time-dependent manner. In light-induced chloroplast development, a key process was the removal of hydrogen peroxide, in which thylakoid-localized PtotAPX played a major role; light-induced chloroplast development was enhanced in PtotAPX-overexpressing transgenic P. tomentosa callus with lower levels of hydrogen peroxide, but was suppressed in PtotAPX antisense transgenic callus with higher levels of hydrogen peroxide. Moreover, the suppression of light-induced chloroplast development in PtotAPX antisense transgenic callus was relieved by the exogenous reactive oxygen species scavenging agent N,N′-dimethylthiourea (DMTU). Based on these results, we propose that PtotAPX-mediated removal of reactive oxygen species plays a key role in chloroplast development from proplastids upon exposure to light in P. tomentosa.

Chloroplast Thylakoidal Ascorbate Peroxidase, PtotAPX, Has Enhanced Resistance to Oxidative Stress in Populus tomentosa

“…Populus tomentosa was used as a donor for genetic transformation and as the wild-type control. The WT and transgenic plants were grown on solid 1/2 Murashige and Skoog (MS) medium supplied with 1.5% ( w / v ) sucrose, 100 mg/L inositol, 0.1mg/L NAA, and 6 g/L agar, pH 5.8, in a growth chamber with a 14 h light/10 h dark photoperiod at 25 °C [ 15 ] and grown for two months. Subsequently, the 2-month-old wild-type and transgenic plants were exposed to 100 μM MV for 5 h to induce oxidative stress.…”

“…The relative expression level of each gene was calculated by the 2 −∆∆Ct method, using the ∆Ct of WT as 1. The expression of PtotAPX in wild-type and transgenic plants was assessed by qRT-PCR using the qRT- PtotAPX -F/R primers described by Li et al [ 15 ]. The P. tomentasa Actin gene was used as an internal control for normalization.…”

“…In order to better clarify the physiological function of the tAPX regulating of ROS levels, in previous studies, we generated overexpressing or antisense PtotAPX transgenic P. tomentosa lines [ 15 ]. Additionally, in this study, we investigated the physiological function of chloroplast PtotAPX in the regulation of H 2 O 2 levels and protection against oxidative stress in woody plants using P. tomentosa as a model plant.…”

Table_2.xls

“…There were three biological replicates for each sample. We performed transcriptome sequencing according to the previous study (Li et al, 2021). Raw RNA-seq reads were available at the NCBI Sequence Read Archive (SRA) under the accession number PRJNA722127.…”

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“…Eight genes including NAC and MYB TFs (PtoWND1A, PtoWND1B, PtoNAM3, PtoNAC3.2, PtoMYB21, PtoMYB23, PtoMYB61.2, and PtoMYB125) were validated, and the primers are shown in Supplementary Table 6. The PCR conditions and processes followed those of Li et al (2021). The P. tomentosa actin gene was used as the internal control.…”