Molecular aspects of plants with altered chloroplast morphology

Chloroplast division involves the coordinated action of multiple proteins that regulate the assembly, positioning and contraction of a stromal Z-ring, inner- & outer-plastid division rings, and a dynamin-related division ring. Different division ring abnormalities result in different chloroplast morphologies that can be detrimental to the plant. Until recently, studies have primarily focused on single chloroplast morphology, the macro chloroplast, to investigate plant developmental abnormalities observed with chloroplast morphology changes. This phenotype appears to have little effect on plant development, despite significantly decreasing the number of chloroplast-per-cell. However, there are a variety of other chloroplast morphologies that are a result of abnormal plastid division, including mini chloroplast morphologies that are more associated with detrimental plant phenotypes. Little is known about how chloroplast morphology and changes in chloroplast number affects plant developmental processes.

To investigate how changes in chloroplast morphology and size effect plant developmental processes, a variety of aberrant chloroplast morphologies were generated in transgenic plants using one of two division gene sequences: ftsZ (EU684588) and minD (EU684589). Plants with changes in chloroplasts were generated in the model plant species, Nicotiana tabacum, and an important commercial cultivar, Brassica oleracea var. botrytis. Investigations found that when plastid division was altered via additional copies of the minD transgene, the generation of macro chloroplasts it did not inhibit cellular expansion but did stunt overall plant growth. These chloroplast morphologies conferred honeycomb-shaped thylakoid grana, resembling low-light adapted structures. By contrast, macro chloroplasts that were generated from ftsZ overexpression, did not stunt plant growth, but did show novel hexagon branching of the thylakoid membranes, which resembled pro-thylakoid structures commonly found in etioplasts.

In N. tabacum in which the chloroplast size decreased, because of the formation of mini chloroplasts, plant growth was stunted and eventually terminal for the altered plants. The cause was further characterised at a proteomic-level and was tentatively linked to a series of J-domainrelated peptides associated with division proteins but appeared to be predominately related to stress proteins and auxin-deficient signal pathways. In addition, terminal plants with mini chloroplast morphologies had a decrease in chloroplast gene expression, specifically photosynthetic-related and polymerase subunit genes, and this may in turn have contributed to activation of senescence pathways and inhibited other cellular processes in the altered plants.

To compare the changes in chloroplast morphology observed in N. tabacum, the expression of the minD (EU684589) division gene was examined in B. oleracea var. botrytis. Regenerated plants overexpressed minD, resulting in macro chloroplasts that did not negatively affect plant growth, in contrast to the N. tabacum observations. Transcription of the minD gene was initiated by a novel and native rbcS promoter isolated from B. oleracea var. botrytis, which may have contributed to a beneficial, but dose-dependent expression of the minD gene for macro chloroplasts. In addition, this section of the research developed a new and efficient regeneration protocol for B. oleracea var. botrytis via somatic embryogenesis.