A facile \u3ci\u3eAgrobacterium\u3c/i\u3e‑mediated transformation method for the model unicellular green algae \u3ci\u3eChlamydomonas reinhardtii\u3c/i\u3e

A reliable and simple Agrobacterium-mediated transformation system for the unicellular green algae model organism Chlamydomonas reinhardtii has been developed. The protocol has been successfully employed with both neomycin phosphotransferase II (nptII) and the phleomycin resistance (bleI) genes coupled with the selective agents paromomycin and zeocin, respectively. A set of binary vectors were assembled that carry the selectable marker cassettes under control either of the Rbcs2 alone or fused to the HSP270A leader sequence, PsaD, or ß-tubulin2 promoters. The corresponding T-DNA elements also harbored a cassette with a codon-optimized version of yellow fluorescence protein (YFP) under control of the Rbcs2 promoter in which the YFP open reading frame was interrupted with the first intron of Rbcs2 to prevent expression in Agrobacterium tumefaciens. The resultant binary vectors were introduced into A. tumefaciens strain C58C1/pMP90, and the derived transconjugants were used for transformation studies with the walled C. reinhardtii strain CC124. Estimated transformation frequencies ranged from 0.09 to 2.86 colonies per 106 cells inoculated. Molecular characterizations on a subset of the transgenic lineages revealed that most of the transgenic events harbored single locus insertions. Moreover, sequencing of captured junction fragments about the T-DNA insertion site showed that minimal disruption of the C. reinhardtii genome occurred. However, the transgenic lineages often harbored truncated T-DNA regions within the non-selectable marker gene cassettes

Functional Analysis of Water Stress-Responsive Soybean GmNAC003 and GmNAC004 Transcription Factors in Lateral Root Development in <i>Arabidopsis</i>

<div><p>In <i>Arabidopsis</i>, NAC (NAM, ATAF and CUC) transcription factors have been found to promote lateral root number through the auxin signaling pathway. In the present study, the role of water stress–inducible soybean <i>GmNAC003</i> and <i>GmNAC004</i> genes in the enhancement of lateral root development under water deficit conditions was investigated. Both genes were highly expressed in roots, leaves and flowers of soybean and were strongly induced by water stress and moderately induced by a treatment with abscisic acid (ABA). They showed a slight response to treatment with 2,4-dichlorophenoxyacetic acid (2,4-D). The transgenic <i>Arabidopsis</i> plants overexpressing <i>GmNAC004</i> showed an increase in lateral root number and length under non-stress conditions and maintained higher lateral root number and length under mild water stress conditions compared to the wild-type (WT), while the transgenic plants overexpressing <i>GmNAC003</i> did not show any response. However, LR development of <i>GmNAC004</i> transgenic <i>Arabidopsis</i> plants was not enhanced in the water-stressed compared to the well-watered treatment. In the treatment with ABA, LR density of the <i>GmNAC004</i> transgenic <i>Arabidopsis</i> was less suppressed than that of the WT, suggesting that <i>GmNAC004</i> counteracts ABA-induced inhibition of lateral root development. In the treatment with 2,4-D, lateral root density was enhanced in both <i>GmNAC004</i> transgenic <i>Arabidopsis</i> and WT plants but the promotion was higher in the transgenic plants. Conversely, in the treatment with naphthylphthalamic acid (NPA), lateral root density was inhibited and there was no difference in the phenotype of the <i>GmNAC004</i> transgenic <i>Arabidopsis</i> and WT plants, indicating that auxin is required for the action of <i>GmNAC004</i>. Transcript analysis for a number of known auxin and ABA related genes showed that <i>GmNAC004</i>'s role may suppress ABA signaling but promote auxin signaling to increase lateral root development in the <i>Arabidopsis</i> heterologous system.</p></div

Transgenic <i>Arabidopsis</i> plants overexpressing soybean <i>GmNAC003</i> and <i>GmNAC004</i>.

<p>(A) Expression of the transgenes in 3-week-old transgenic plants quantified by qRT-PCR using <i>Arabidopsis</i> ubiquitin as the reference gene. The tissues were sampled from homozygous transgenic plants having a single insertion. Error bars are the standard errors of the means from three samples of ten plants. (B) Growth of the 4-week old and T4 generation <i>GmNAC003</i> (events N3.09 and N3.10) and <i>GmNAC004</i> transgenic (events N4.01 and N4.03) and WT plants.</p

Relative transcript abundance of <i>GmNAC003</i> and <i>GmNAC004</i> in response to water stress.

<p>Transcript abundance was quantified using qRT-PCR and the data were normalized to the four best internal control genes (<i>CYP2, IDE, UNK1</i> and <i>UNK2</i>) based on the M stability of GeNorm analysis. Mean relative expression levels were transformed to a value of 1 for the sample having lowest expression. Error bars are standard errors of the means from three replications. (A) Response to water deficit stress. Plants at the 2-leaf (V2) growth stage were allowed to grow without supply of additional water until desired stem water potentials were achieved. V2, V3 and V6 are the growth stages of the control plants corresponding to the time when water stressed-plants were sampled. (B) Response to dehydration stress. V2 growth stage plants were harvested and allowed to dehydrate in a growth chamber for the designated times.</p

Representative root growth of transgenic <i>Arabidopsis</i> plants overexpressing <i>GmNAC004</i> in response to water deficit conditions.

<p>Two T4 homozygous transgenic <i>Arabidopsis</i> (N-4.01 and N-4.03) and WT plants were grown on nutrient agar plates diffused with different concentrations of PEG. The plants were 12 days old (8 days after stress exposure).</p

Role of GmNAC004 in regulation of lateral root density of transgenic <i>Arabidopsis</i> plants.

<p>(A) Transgenic <i>Arabidopsis</i> overexpressing <i>GmNAC004</i> in response to ABA and 2,4-D treatments. Four-day-old seedlings of WT and <i>GmNAC004</i> transgenic plants (T4 generation) were exposed to 5 µM ABA, 20 nM 2,4-D, 2 µM NPA or their combinations for 7 days. Control treatment was not treated with hormones. Error bars are the standard errors of the means from six replications. Asterisks denote significant differences at 95% between the WT and transgenic plants. (B) Expression of representative ABA and auxin signaling genes in transgenic GmNAC003 and GmNAC004 <i>Arabidopsis</i> plants. Duncan multiple-mean comparisons were used and different letters indicates differences of the means.</p