Transgenic tobacco overexpressing Brassica juncea HMG-CoA synthase 1 shows increased plant growth, pod size and seed yield

Abstract

Seeds are very important not only in the life cycle of the plant but they represent food sources for man and animals. We report herein a mutant of 3-hydroxy-3-methylglutaryl-coenzyme A synthase (HMGS), the second enzyme in the mevalonate (MVA) pathway that can improve seed yield when overexpressed in a phylogenetically distant species. In Brassica juncea, the characterisation of four isogenes encoding HMGS has been previously reported. Enzyme kinetics on recombinant wild-type (wt) and mutant BjHMGS1 had revealed that S359A displayed a 10-fold higher enzyme activity. The overexpression of wt and mutant (S359A) BjHMGS1 in Arabidopsis had up-regulated several genes in sterol biosynthesis, increasing sterol content. To quickly assess the effects of BjHMGS1 overexpression in a phylogenetically more distant species beyond the Brassicaceae, wt and mutant (S359A) BjHMGS1 were expressed in tobacco (Nicotiana tabacum L. cv. Xanthi) of the family Solanaceae. New observations on tobacco OEs not previously reported for Arabidopsis OEs included: (i) phenotypic changes in enhanced plant growth, pod size and seed yield (more significant in OE-S359A than OE-wtBjHMGS1) in comparison to vector-transformed tobacco, (ii) higher NtSQS expression and sterol content in OE-S359A than OE-wtBjHMGS1 corresponding to greater increase in growth and seed yield, and (iii) induction of NtIPPI2 and NtGGPPS2 and downregulation of NtIPPI1, NtGGPPS1, NtGGPPS3 and NtGGPPS4. Resembling Arabidopsis HMGS-OEs, tobacco HMGS-OEs displayed an enhanced expression of NtHMGR1, NtSMT1-2, NtSMT2-1, NtSMT2-2 and NtCYP85A1. Overall, increased growth, pod size and seed yield in tobacco HMGS-OEs were attributed to the up-regulation of native NtHMGR1, NtIPPI2, NtSQS, NtSMT1-2, NtSMT2-1, NtSMT2-2 and NtCYP85A1. Hence, S359A has potential in agriculture not only in improving phytosterol content but also seed yield, which may be desirable in food crops. This work further demonstrates HMGS function in plant reproduction that is reminiscent to reduced fertility of hmgs RNAi lines in let-7 mutants of Caenorhabditis elegans.

Figure 1. Outline of isoprenoid biosynthesis pathways in plants.

Enzymes are shown in bold. Pathway inside the mitochondria and plastid are boxed. Arrows between cytosolic and plastid compartments represent metabolic flow between them (greater arrow for more flux). Abbreviations: ABA, abscisic acid; AACT, acetoacetyl-CoA thiolase; BR6OX2, brassinosteroid-6-oxidase 2; CYP710A1, sterol C-22 desaturase; CYP85A1, cytochrome P450 monooxygenase; DMAPP, dimethylallyl diphosphate; DWF1, delta-24 sterol reductase; DXR, 1-deoxy-D-xylulose 5-phosphate reductoisomerase; DXS, 1-deoxy-D-xylulose 5-phosphate synthase; FPP, farnesyl diphosphate; GA-3-P, glyceraldehyde-3-phosphate; FPPS, farnesyl diphosphate synthase; GAs, gibberellins; GGPP, geranylgeranyl diphosphate; GGPPS, geranylgeranyl diphosphate synthase; GPP, geranyl diphosphate; HMG-CoA, 3-hydroxy-3-methylglutaryl-CoA; HMGS, 3-hydroxy-3-methylglutaryl-CoA synthase; HMGR, 3-hydroxy-3-methylglutaryl-CoA reductase; IPP, isopentenyl diphosphate; IPPI, isopentenyl/dimethylallyl diphosphate isomerase; Q₁₀, coenzyme Q₁₀; SMT, sterol methyltransferase; SQS, squalene synthase. HMGS is marked in red colour. The expression levels of enzymes analysed in this work are marked in blue colour.

Figure 2. Molecular analysis of representative transgenic tobacco HMGS-OEs.

(A) Western blot analysis using antibodies against BjHMGS1 to verify the expression of BjHMGS1 (52.4-kDa) in representative vector (pSa13)-transformed control and HMGS-OEs (OE-wtBjHMGS1 and OE-S359A). Putative tobacco HMGS-OEs were designated as OE-wtBjHMGS1 (lines “401”, “402” and “404”) and OE-S359A (lines “602”, “603” and “606”). Bottom, Coomassie Blue-stained gel of total protein loaded (20 µg per well). Three independent lines per construct were analysed. (B) Northern blot analysis of BjHMGS1 in representative vector (pSa13)-transformed control and HMGS-OEs. The expected 1.7-kb BjHMGS1 band is marked with an arrowhead. Bottom gels show rRNA (20 µg per lane). Two independent lines per construct are shown. The two independent lines of OE-wtBjHMGS1 plants labelled “401” and “402”, and two independent lines of OE-S359A plants labelled “603” and “606” used in further tests are underlined.

Figure 3. Comparison in growth between tobacco HMGS-OE seedlings/plants and vector-transformed control.

(A) Seedlings 14-d post-germination. The vector-transformed control is labelled “pSa13”, two independent lines of OE-wtBjHMGS1 plants are labelled “401” (two representative seedlings of this OE construct were shown) and “402” (three representative seedlings of this OE construct were shown) and two independent lines of OE-S359A plants are labelled “603” (two representative seedlings of this OE construct were shown) and “606” (three representative seedlings of this OE construct were shown). Bar  = 1 cm. (B) Root length measurements of 14-d-old seedlings showed that tobacco HMGS-OE roots grow faster than the vector (pSa13)-transformed control. Values are mean ±SD (n = 30); Bars are SD. (C) Dry weight determination of 14-d-old seedlings shows that tobacco HMGS-OEs possess a higher mass than the vector-transformed control. Values are mean ± SD (n = 30); Bars are SD. (D) Representative greenhouse-grown plants photographed 80-d after germination. OE plants are labelled OE-wtBjHMGS1 and OE-S359A. Two independent lines of OE-wtBjHMGS1 plants, “401” (upper) and “402” (lower) and two independent lines of OE-S359A plants, “603” (upper) and “606” (lower) are shown. Bar  = 10 cm. (E) Statistical analysis on height of 80-d-old transgenic plants. Values are mean ±SD (n = 6); Bars are SD; H, higher than control; a indicates significant difference between HMGS-OE and the vector (pSa13)-transformed control (P<0.01 by the Student's t-test); b indicates significant difference between OE-wtBjHMGS1 and OE-S359A (P<0.01 by the Student's t-test). pSa13, vector-transformed control; two independent lines of OE-wtBjHMGS1 (“401” and “402”) and two independent lines of OE-S359A (“603” and “606”) were used for growth rate measurement.

Figure 4. Comparison in plant growth between 98-d-old greenhouse-grown HMGS-OEs and vector-transformed tobacco.

(A) Representative plants photographed 98-d after germination show differences in growth between HMGS-OE tobacco plants and vector-transformed control. Bar  = 10 cm. (B) Analysis on height of 98-d-old transgenic plants. (C) Representative tobacco leaves photographed 98-d after germination with growth differences between HMGS-OE and vector-transformed tobacco. Bar  = 10 cm. (D) Analysis on fresh weight, length and width of bottom-most four leaves from a 98-d-old tobacco plant. Values are mean ± SD (n = 6); Bars are SD; **, P<0.01; *, P<0.05; ** and *, significantly higher than control, by the Student's t-test. The vector-transformed control is labelled “pSa13”, three independent lines of OE-wtBjHMGS1 plants are labelled “401”, “402” and “404”, and three independent lines of OE-S359A plants are labelled “602”, “603” and “606”.

Figure 5. Comparison in plant growth between 210-d-old greenhouse-grown HMGS-OEs and vector-transformed tobacco.

(A) Representative plants photographed 210-d after germination show differences in growth between HMGS-OE tobacco plants and the vector (pSa13)-transformed control. Bar  = 10 cm. (B) Analysis on height of 210-d-old transgenic plants. Values are mean ± SD (n = 6); Bars are SD; **, P<0.01; *, P<0.05; ** and *, significantly higher than control, by the Student's t-test. The vector-transformed control is labelled “pSa13”, three independent lines of OE-wtBjHMGS1 plants are labelled “401”, “402” and “404”, and three independent lines of OE-S359A plants are labelled “602”, “603” and “606”.

Figure 6. Tobacco HMGS-OEs show increased seed yield.

(A) Phenotype of tobacco pods. pSa13, vector-transformed control; “401” and “402”, two independent lines of OE-wtBjHMGS1 and “603” and “606”, two independent lines of OE-S359A. Scale bar  = 1 cm. (B) Total dry weight of 30 tobacco pods. (C) Average dry weight per pod. (D) Total dry weight of seeds from 30 pods. (E) Total seed number per 30 pods. (F) Average seed number per pod. (G) Average dry weight of 100 seeds in control and HMGS-OEs. Thirty independent readings were taken for each line. Values are means ± SD, n = 30. a indicates significant difference between HMGS-OE and the vector (pSa13)-transformed control; b indicates significant difference between OE-wtBjHMGS1 and OE-S359A. H, value higher than the control (P<0.05 or 0.01 by the Student's t-test).

Figure 7. Expression of HMGS downstream genes by qRT-PCR in 20-d-old tobacco seedlings of HMGS-OEs.

Total RNA was extracted from 20-d-old tobacco seedlings of vector (pSa13)-transformed control, three independent lines of OE-wtBjHMGS1 (lines “401”, “402” and “404”) and three independent lines of OE-S359A (lines “602”, “603” and “606”). H, value higher than the control (P<0.05, Student's t-test); L, value lower than the control (P<0.05, Student's t-test). Values are means ±SD (n = 3). a indicates significant difference between HMGS-OE and the vector (pSa13)-transformed control for at least two independent lines from three independent lines; b indicates significant difference between OE-wtBjHMGS1 and OE-S359A for at least two independent lines from three independent lines.

Figure 8. Expression of plastidial GGPPSs determined by qRT-PCR in 20-d-old tobacco seedlings of HMGS-OEs.

Total RNA was extracted from 20-d-old tobacco seedlings of vector (pSa13)-transformed control, three independent lines of OE-wtBjHMGS1 (lines “401”, “402” and “404”) and three independent lines of OE-S359A (lines “602”, “603” and “606”). H, value higher than the control (P<0.05, Student's t-test); L, value lower than the control (P<0.05, Student's t-test). Values are means ± SD (n = 3).