Individual maize chromosomes in the C(3) plant oat can increase bundle sheath cell size and vein density

Abstract

C(4) photosynthesis has evolved in at least 66 lineages within the angiosperms and involves alterations to the biochemistry, cell biology, and development of leaves. The characteristic "Kranz" anatomy of most C(4) leaves was discovered in the 1890s, but the genetic basis of these traits remains poorly defined. Oat × maize addition lines allow the effects of individual maize (Zea mays; C(4)) chromosomes to be investigated in an oat (Avena sativa; C(3)) genetic background. Here, we have determined the extent to which maize chromosomes can introduce C(4) characteristics into oat and have associated any C(4)-like changes with specific maize chromosomes. While there is no indication of a simultaneous change to C(4) biochemistry, leaf anatomy, and ultrastructure in any of the oat × maize addition lines, the C(3) oat leaf can be modified at multiple levels. Maize genes encoding phosphoenolpyruvate carboxylase, pyruvate, orthophosphate dikinase, and the 2'-oxoglutarate/malate transporter are expressed in oat and generate transcripts of the correct size. Three maize chromosomes independently cause increases in vein density, and maize chromosome 3 results in larger bundle sheath cells with increased cell wall lipid deposition in oat leaves. These data provide proof of principle that aspects of C(4) biology could be integrated into leaves of C(3) crops.

Figure 1.

Alterations to leaf morphology in OMA lines. A and B, Distance between adjacent veins (A) and number of palisade M cells separating adjacent vascular bundles (B). C and D, PBS cell cross-sectional area (C) and PBS-M cross-sectional area ratio (D) in maize, oat, and OMA leaves. Results are expressed as means ± se of three biological replicates with a minimum of 10 measurements per leaf. Starred results are statistically significant relative to oat controls (P < 0.01).

Figure 2.

Maize chromosome 3 causes lipid deposition in oat PBS cell walls. Fluorescence (A–C) and TEM (D–F) images of transverse sections of maize (A and D), oat (sun II; B and E), and OMA 3.01 (C and F) leaves are shown. Lipid detection was carried out by staining sections with Nile red (A–C). Suberin detection was by TEM (D–F). Asterisks indicate MS. In TEM images, arrows indicate suberin layers. Bars = 20 μm (A–C) and 500 nm (D–F).

Figure 3.

Photosynthetic assimilation rates are largely unchanged by maize chromosomal introduction. Light-saturated photosynthetic assimilation rates (A) over a range of leaf intercellular CO₂ concentrations (Ci) for leaves of maize, oat, and OMA lines are shown. Each panel represents mean assimilation rates of maize and wild-type oat or chromosomal means of OMA lines. Results are expressed as means of at least three biological replicates ± se. Measurements were taken at 25°C, leaf vapor pressure deficit of less than 1.5 kPa, and PFD of 1,500 μmol m⁻² s⁻¹.

Figure 4.

Photosynthetic light responses are largely unchanged by maize chromosomal introduction. Photosynthetic assimilation rates (A) over a range of light intensities at ambient CO₂ for leaves of maize, oat, and OMA lines are shown. Each panel represents mean assimilation rates of maize and wild-type oat or chromosomal means of OMA lines. Results are expressed as means of at least three biological replicates ± se. Measurements were taken at 25°C, leaf vapor pressure deficit of less than 1.5 kPa, and [CO₂] of 380 µL L^–1.

Figure 5.

Detection of maize-derived transcripts associated with C₄ photosynthesis in leaves of OMA plants. Total RNA was isolated from mature leaves of OMA plants and resolved on a 1.2% agarose-formaldehyde gel. RNA (10 µg per lane) was hybridized with a radiolabeled maize probe for the transcript of interest. RNA from maize, oat, and an OMA line with a different maize chromosome were probed as controls. rRNA (25S) from the gel used for blotting stained with ethidium bromide is shown as a loading control below each blot. Transcripts detected with maize-specific ZmCA2 (2.0 kb), ZmDCT2 (1.9 kb), ZmMDH6 (1.5 kb), ZmDCT1 (2.1 kb), ZmME1 (2.2 kb), ZmPPDK (3.5 kb), ZmPEPC1 (3.2 kb), and ZmOMT1 (1.9 kb) probes were of the expected sizes.

Figure 6.

Thylakoid stacking in grana of oat PBS cells is unchanged by maize chromosomal introduction. Numbers of thylakoid stacks per granum in M (A) and bundle sheath chloroplasts (B) in maize, oat, and OMA leaves are shown. Results are expressed as means ± se of three biological replicates with a minimum of 10 measurements per leaf. Starred results are statistically significant relative to oat controls (P < 0.01).