Tissue specific protochlorophyll(ide) forms in dark-forced shoots of grapevine (Vitis vinifera L.)

Cuttings of grapevine (Vitis vinifera L. cv. Chardonnay) were dark-forced at least three weeks. Pigment contents, 77 K fluorescence emission and excitation spectra of the leaves, petioles and stems and transmission electron micrographs of the etioplasts from leaves and the chlorenchyma tissues of the stems were analysed. The dark-grown leaves and stems contained 8 to 10 and 3 to 5 mug/g fresh weight protochlorophyllide and its esters, respectively. HPLC analysis showed that the molar ratio of the unesterified and esterified pigments was 7:3 in the shoot developed in darkness. The dark-forced leaves contained carotenoids identified as: neoxanthin, violaxanthin, antheraxanthin, lutein and beta-carotene. Detailed analyses of the fluorescence spectra proved that all tissues of the dark-forced shoots had protochlorophyllide or protochlorophyll forms with emission maxima at 628, 636, 644, 655 and 669 nm. The 628 and 636 nm emitting forms were present in all parts of the dark-forced shoot, but dominated in the stems, which may indicate an organ specificity of the etioplast development. Variations in the distribution of the pigment forms were even found in the different tissues of the stem. The subepidermal layers were more abundant in the 655 nm form than the parenchyma cells of the inner part of the cortex and the pith. In the latter cells, the plastid differentiation stopped in intermediary stages between proplastids and etioplasts. The plastids in the subepidermal layers had developed prolamellar body structures, which were similar to those of etiolated leaves. The results highlight the importance of organ- and tissue specificity of plastid differentiation for chlorophyll biosynthesis and greening of different plant organs.