Variation Analysis of Physiological Traits in Betula platyphylla Overexpressing TaLEA-ThbZIP Gene under Salt Stress

Abstract

The aim of this study was to determine whether transgenic birch (Betula platyphylla) ectopic overexpressing a late embryogenesis abundant (LEA) gene and a basic leucine zipper (bZIP) gene from the salt-tolerant genus Tamarix (salt cedar) show increased tolerance to salt (NaCl) stress. Co-transfer of TaLEA and ThbZIP in birch under the control of two independent CaMV 35S promoters significantly enhanced salt stress. PCR and northern blot analyses indicated that the two genes were ectopically overexpressed in several dual-gene transgenic birch lines. We compared the effects of salt stress among three transgenic birch lines (L-4, L-5, and L-8) and wild type (WT). In all lines, the net photosynthesis values were higher before salt stress treatment than afterwards. After the salt stress treatment, the transgenic lines L-4 and L-8 showed higher values for photosynthetic traits, chlorophyll fluorescence, peroxidase and superoxide dismutase activities, and lower malondialdehyde and Na+ contents, compared with those in WT and L-5. These different responses to salt stress suggested that the transcriptional level of the TaLEA and ThbZIP genes differed among the transgenic lines, resulting in a variety of genetic and phenotypic effects. The results of this research can provide a theoretical basis for the genetic engineering of salt-tolerant trees.

Fig 1. Map of the T-DNA construct and identification of overexpressing TaLEA and ThbZIP transgenic birches with PCR.

(A) Schematic of the T-DNA region of the binary vector pROKII-TaLEA-ThbZIP. RB, right border; Pnos, nopaline synthase promoter; NptII, kanamycin resistance gene; Tnos, nopaline synthase terminator; P35S, CaMV 35S promoter; Sma I, Sac I, BamH I, and Kpn I, four different restriction enzyme sites; TaLEA, TaLEA gene; ThbZIP, ThbZIP gene; LB, left border. Agarose gel electrophoresis of PCR products from wild type and transgenic lines with the primer of NptII (B), TaLEA (C), ThbZIP (D). M, DNA marker; PC, positive control; 1–11, eleven Km resistant lines; L-4, 5, 7, 8, 9, five transgenic lines both containing TaLEA and ThbZIP genes; WT, wild type plantlet; H₂O, double-distilled water as negative control.

Fig 2. Regeneration of overexpressing TaLEA and ThbZIP transgenic birch.

(A) The transgenic callus (red circle) have formed on one of the cut sites in a leaf segment. (B) Close-up view of the circled area in (A). (C) Some transgenic calls gained from leaf segments. (D) transgenic cluster of shoots has formed from a callus. (E) Four transgenic shoots were transferred to rooting medium. (F) One-month-old transgenic plants were grown in sterile soil media. (G) Three-month-old transgenic plants were grown in a greenhouse.

Fig 3. Northern blot analysis of TaLEA and ThbZIP expression in transgenic birches.

Total RNA was extracted from the aerial tissues of one-month-old wild type and transgenic lines. The full length TaLEA and ThbZIP genes labeled with DIG-dUTP were used as probes. rRNA, ribosomal RNAs from different samples; TaLEA, target band of TaLEA gene; ThbZIP, target band of ThbZIP gene; L-4, 5, 8, three transgenic lines both containing TaLEA and ThbZIP genes; WT, wild type plantlet, respectively.

Fig 4. NaCl stress-tolerance test of transgenic birch ectopic overexpressing TaLEA and ThbZIP.

Stems of transgenic and wild type plants were cultured on WPM medium containing 2 g L^-1 (A), 4 g L^-1 (B) or 6 g L^-1 NaCl (C). Shoots of transgenic and wild type plants were transferred to 1/2 MS root medium containing 4 g L^-1 NaCl (D), or 6 g L^-1 NaCl (E). Photographs were taken 20 d following stress treatment. WT, wild type; L-4, transgenic line 4; L-8, transgenic line 8.

Fig 5. The phenotypes of WT on day 8 under different concentrations of NaCl.

From left to right were WT, the treatment of 2 g L^-1, 4 g L^-1, 6 g L^-1 and 8 g L^-1 of salt stress, respectively.

Fig 6. Pn values of WT under different concentrations salt stress.

Fig 7. F_v/F_m values of WT under different concentrations salt stress.

Fig 8

Pn-PPFD and Pn-Ca curves of different transgenic lines before stress (A, and C) and 8 d after salt stress (B and D).

Fig 9

Pn (A), Gs (B), Ci (C) and Tr (D) values of different lines after different days salt stress.

Fig 10. Fv/Fm values of different lines after different days salt stress.

Fig 11

POD (A), SOD (B), MDA (C) and Na⁺ (D) values of different lines after different days salt stress.