Protochlorophylls in Cucurbitaceae - Distribution, biosynthesis and phylogeny Author Kruk, Jerzy Author Gabruk, Michał Author Kryszczak, Marian Author Wąsik, Piotr text Phytochemistry 2022 113110 2022-05-31 197 1 8 http://dx.doi.org/10.1016/j.phytochem.2022.113110 journal article 10.1016/j.phytochem.2022.113110 1873-3700 8235034 2.2. Pigment synthesis in Cucurbita seedlings An analysis of porphyrin pigments in etiolated C. pepo ‘Miranda’ seedlings revealed that when subjected to continuous illumination ( Fig. 4 ), Chl a content in the control plants increased gradually, as expected, while the level of Chl-GG, a precursor of Chl a , was low and did not change significantly during illumination, like that of Pchl-GG. However, when the seedlings were incubated with ALA (precursor of the porphyrin ring) before illumination, the Chl-GG content increased dramatically with illumination time, while the synthesis of Chl a was inhibited. The synthesis of Pchl-GG was not affected very much ( Fig. 4 ). It is supposed that Chl a is synthesized by Chl synthase from Chlide a and both phytyl-PP or GG-PP, depending on the plant species, light conditions (darkness or light), the age of the plants and other factors ( Schoefs et al., 1998 ; Schoefs and Bertrand 2000 , Schmid et al., 2001 and 2002 ). n.d. – not determined. These data suggest that Chl a is not synthesized from GG-PP via Chl-GG but rather directly from phytyl-PP in C. pepo seedlings. This conclusion is supported by the lack of other Chl a precursors (Chl-DHGG, Chl-THGG) which are intermediates in the pathway from Chl-GG to Chl a (data not shown). The observation that the Chl a level did not increase after ALA feeding suggests that the phytyl-PP supply is limited. An analysis of the composition of Pchls in primary leaves during illumination ( Fig. 5 ) shows that Pchl-GG in the C. pepo ‘Miranda’ control plants was the dominating species and the content of individual compounds did not change very much during illumination, apart from Pchl-DHGG whose contribution increased fourfold. In the presence of ALA, a gradual, but not very pronounced, increase in Pchl-GG and Pchl-DHGG levels occurred at the expense of the two other Pchls. These data suggest that the synthesis of Pchls in the leaves of developing seedlings is a relatively slow process, but not limited by the supply of substrates, such as Pchlide ( Fig. 4 ). It is also probable that the enzyme is only active at the very early stages of the seedlings’ development and its expression decreases quickly with the age of the seedlings. Fig. 1. (a) Two-dimensional HPLC chromatogram of protochlorophylls extracted from the seed coat of pumpkin ( Cucurbita pepo ) seeds and (b) the corresponding one-dimensional chromatogram using absorption detection at 435 nm. Analysis was performed using C30 YMC column, methanol/ethyl acetate (34/16) as eluent and 1.5 ml/min flow. Table 1 Absorption maxima of porphyrins determined in this study and reported in the literature.

Compound	Absorption maxima (nm)	Solvent	Reference
MV-Pchls	432, 627	methanol/ethyl acetate, 34/16 (v/v)	this study
MV-Pchl	432, 622	diethyl ether	Houssier and Sauer (1969) ,
MV-Pchl	432, 623	diethyl ether	Ellsworth and Nowak (1974)
MV-Pchlide	433, 627	methanol/ethyl acetate, 34/16 (v/v)	this study
MV-Pchlide	432, 622.5	diethyl ether	Kruk and My´sliwa-Kurdziel (2004)
DV-Pchls	438, 628	methanol/ethyl acetate, 34/16 (v/v)	this study
DV-Pchl	438, 622	diethyl ether	Stanier and Smith (1959)
DV-Pchl	437, 622	diethyl ether	Houssier and Sauer (1969)
DV-Pchl	437, 623	acetonitrile/acetone, 55/45 (v/v)	Mukaida et al. (1993)
DV-Pchl	436, 622	diethyl ether	Mukaida and Nishikawa (1990)
DV-Pchlide	437, 627.5	methanol/ethyl acetate, 34/16 (v/v)	this study
DV-Pchlide	436.5, 623	diethyl ether	Kruk and My´sliwa-Kurdziel (2004)
MV-Ppheos	418, n.d.*	methanol/acetonitrile, 1:1 (v/v)	this study
MV-Ppheo	417, 567, 588	acetonitrile/acetone, 55/45 (v/v)	Mukaida et al. (1993)
MV-Ppheo	418, 565, 588	diethyl ether	Houssier and Sauer (1969)
MV-Ppheophorbide	418, 563, 597.5	methanol/acetonitrile, 1:1 (v/v)	this study
DV-Ppheos	422, n.d.	methanol/acetonitrile, 1:1 (v/v)	this study
DV-Ppheo	421, 569, 591	acetonitrile/acetone, 55/45 (v/v)	Mukaida et al. (1993)
DV-Ppheo	421, 566, 587	diethyl ether	Mukaida and Nishikawa (1990)
DV-Ppheo	421.5, 567, 591	diethyl ether	Jones (1966)
DV-Ppheophorbide	421, 566.5, 597.5	methanol/acetonitrile, 1:1 (v/v)	this study

Table 2 Composition of protochlorophylls in the seed coats of the species and varieties of Cucurbitaceae under investigation. The compounds are ordered according to increasing retention times in HPLC chromatograms. The data are the means of 3 replicates with SE ≤ 20% of the mean values.

Protochlorophylls (% total)	MV-GG	MV-DHGG	MV-THGG	MV-phytol	DV-GG	DV-DHGG	DV-THGG	DV-phytol
Log k’	1.204	1.234	1.273	1.335	1.396	1.425	1.463	1.528
Cucurbita pepo	11.3	10.8	10.6	8.3	16.7	12.5	11.7	17.9
Cucurbita maxima	14.9	12.7	15.7	2.2	20.5	13.9	17.8	2.2
‘Melon Yellow’ Cucurbita maxima a	9.6	9.5	13.3	0.6	21.9	18.4	25.2	1.4
Marrow	7.5	4.5	15.7	18.4	8.4	3.0	17.9	24.1
Pattypan squash	1.0	1.6	5.6	9.4	6.9	7.1	25.3	43.1
Echinocystis lobata a	8.7	2.2	15.2	64.4	3.4	0.4	1.8	3.8
Luffa cylindrica a	23.6	3.7	4.2	68.6	0	0	0	0
Cyclanthera brachystachys	0.2	0	1.1	89.3	0.1	0	0.7	8.6

a Fresh seeds. When an analogous experiment was performed with a different species, C. maxima ‘Bambino’, the pigment content changes were similar to those for C. pepo ‘Miranda’ in the control plants, i.e., Chl a content increased gradually, while that of Chl-GG and Pchl-GG did not change significantly during illumination ( Fig. 6 ). The content of individual Pchls was dominated by Pchl-GG and did not change very much during illumination ( Fig. 5 ). Next, we tried to follow Pchl synthase activity using different preparations, namely homogenates of seed coats, the inner seed coats of C. maxima ‘Bambino’, prolammelar bodies (PLB) isolated from etiolated leaves of C. maxima ‘Bambino’ and homogenates of etiolated leaves of C. pepo ‘Miranda’. Different isolation buffers and additional preparation treatments, such as repeated freeze-thawing, were also used. We applied a broad substrate concentration range of both Pchlide and side-chain precursors (GG-PP and phytol-PP) and incubation periods (see Experimental section). However, in none of the preparations was Pchl synthase activity found. This suggests the latency of the synthase and/or that the reaction rate of the enzyme is slow, like what was demonstrated in the case of Chl synthase ( Lindsten et al., 1990 ). It is also possible that in seeds the enzyme is active only at the early stages of seed development which were not analyzed in our case. Nevertheless, the homogenates of etiolated leaves of C. pepo ‘Miranda’ were active in Chl-GG and Chl-phytol synthesis, using Chlide and appropriate side-chain substrates ( Fig. S3 ), indicating that the assay method, at least for the chlorophyll synthase activity, is appropriate. Both Chl-GG and Chl-phytol were found as double peaks, showing identical absorption maxima to those of Chl a , and result probably from the presence of both cis/trans isomers of the side-chain substrates.