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Chloroplast division and morphology are differentially affected by overexpression of FtsZ1 and FtsZ2 genes in Arabidopsis - PubMed

Chloroplast division and morphology are differentially affected by overexpression of FtsZ1 and FtsZ2 genes in Arabidopsis

K D Stokes et al. Plant Physiol. 2000 Dec.

Abstract

In higher plants, two nuclear gene families, FtsZ1 and FtsZ2, encode homologs of the bacterial protein FtsZ, a key component of the prokaryotic cell division machinery. We previously demonstrated that members of both gene families are essential for plastid division, but are functionally distinct. To further explore differences between FtsZ1 and FtsZ2 proteins we investigated the phenotypes of transgenic plants overexpressing AtFtsZ1-1 or AtFtsZ2-1, Arabidopsis members of the FtsZ1 and FtsZ2 families, respectively. Increasing the level of AtFtsZ1-1 protein as little as 3-fold inhibited chloroplast division. Plants with the most severe plastid division defects had 13- to 26-fold increases in AtFtsZ1-1 levels over wild type, and some of these also exhibited a novel chloroplast morphology. Quantitative immunoblotting revealed a correlation between the degree of plastid division inhibition and the extent to which the AtFtsZ1-1 protein level was elevated. In contrast, expression of an AtFtsZ2-1 sense transgene had no obvious effect on plastid division or morphology, though AtFtsZ2-1 protein levels were elevated only slightly over wild-type levels. This may indicate that AtFtsZ2-1 accumulation is more tightly regulated than that of AtFtsZ1-1. Plants expressing the AtFtsZ2-1 transgene did accumulate a form of the protein smaller than those detected in wild-type plants. AtFtsZ2-1 levels were unaffected by increased or decreased accumulation of AtFtsZ1-1 and vice versa, suggesting that the levels of these two plastid division proteins are regulated independently. Taken together, our results provide additional evidence for the functional divergence of the FtsZ1 and FtsZ2 plant gene families.

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Figures

Figure 1
Figure 1

Specificity of AtFtsZ antipeptide antibodies. A, Immunoblots of proteins isolated from wild-type Arabidopsis leaf extracts were probed with 1-1A (lanes 1–3) or 2-1A (lanes 4–6) antibodies raised against peptide from AtFtsZ1-1 or AtFtsZ2-1, respectively. Antibodies were preincubated with purified, recombinant AtFtsZ1-1 protein (lanes 2 and 5) or AtFtsZ2-1 protein (lanes 3 and 6). B, Immunoblot of proteins isolated from leaf extracts of wild-type (lanes 1 and 2), AtFtsZ1-1 antisense (lanes 3 and 4), or AtFtsZ2-1 antisense (lanes 5 and 6) plants. Blots were probed with either 1-1A (lanes 1, 3, and 5) or 2-1A (lanes 2, 4, and 6) antibodies. The 46- and 40-kD polypeptides are indicated by markers. Equivalent volumes of plant extracts were loaded in each lane.

Figure 2
Figure 2

Phenotypes of transgenic plants overexpressing AtFtsZ1-1 or AtFtsZ2-1. Mesophyll cells are shown from the first leaves of 23-d-old plants transformed with the empty pART27 vector (A), the AtFtsZ1-1 sense transgene (B–E), or the AtFtsZ2-1 sense transgene (F). Tissue was prepared for imaging with differential interference contrast optics using methods described previously (Pyke and Leech, 1991). Bar = 25 μm in all figures. A three-dimensional rotating reconstruction of cells with phenotypes similar to those shown in C and D can be found in a video supplement at

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Figure 3
Figure 3

Immunoblot analysis of plant extracts overexpressing AtFtsZ1-1. Proteins in extracts from 23-d-old transgenic plants were separated by SDS-PAGE, transferred to nitrocellulose, and probed with antipeptide antibodies raised against AtFtsZ1-1 (A) or AtFtsZ2-1 (B). Lane 1, Empty vector control (E); lanes 2 through 4, wild type (C); lanes 5 through 10, transgenic plants with wild-type-like (W, lanes 5 and 6), intermediate (I, lane 7), or severe (S, lanes 8–10) phenotypes. Equal loading of all samples was confirmed by staining the membranes with ponceau S (data not shown).

Figure 4
Figure 4

Relative levels of the 40-kD AtFtsZ1-1 polypeptide in plants expressing the AtFtsZ1-1 transgene. Densitometry readings from an immunoblot loaded with increasing amounts of extract from an empty vector control plant (lanes 1–4) were used to construct a standard concentration curve for AtFtsZ1-1. AtFtsZ1-1 levels in the other plant extracts (lanes 5–13), all loaded so that the densitometry reading produced by the 40-kD AtFtsZ1-1 polypeptide on immunoblots fell within the linear range of the standard curve, were then calculated, taking into account the volume loaded. The volume loaded, signal produced on immunoblots, and calculated level of AtFtsZ1-1 relative to that in the empty-vector controls are shown for three Columbia wild type plants (lanes 5–7), and transgenic plants with wild-type-like (WTL, lanes 8 and 9), intermediate (INT, lane 10), or severe (SEVERE, lanes 11–13) phenotypes.

Figure 5
Figure 5

Immunoblot analysis of transgenic plant extracts expressing the AtFtsZ2-1 transgene. Proteins in extracts from 23-d-old transgenic plants were separated by SDS-PAGE, transferred to nitrocellulose, and probed with antipeptide antibodies raised against AtFtsZ2-1 (A) or AtFtsZ1-1 (B). Lane 1, Empty vector control (E); lane 2, wild type (C); lanes 3 through 18, T1 or T3 transgenic plants with wild-type-like (W, lanes, 3, 4, 6–9, 11, 13, and 16–18), intermediate (I, lanes 10 and 14), or severe (S, lanes 5 and 15) phenotypes. Equal loading of all samples was confirmed by staining the membranes with ponceau S (data not shown).

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