Distribution of carotenoids and Y1 mRNA in maize kernels
--Brent Buckner and Diane Janick-Buckner

Yellow-kerneled maize is known to contain carotenoids, while white-kerneled maize lines are thought to contain little if any carotenoids. The plant hormone abscisic acid (ABA), which is involved in maintaining kernel dormancy, is derived from carotenoids. Therefore, if white kernels contain no carotenoid biosynthetic activity the kernels would not contain ABA and, subsequently, would be expected to be viviparous. Viviparous mutants of maize are known, however, kernels that are white as a result of being homozygous for a recessive allele of Y1 are not viviparous. Therefore, we have investigated the distribution of carotenoids and Y1 mRNA in endosperm and embryos that are homozygous for dominant or recessive alleles of Y1.

The standard Y1 line used in these experiments was a hybrid of inbred lines Q66 and Q67. The y1 standard line used was derived from a heterozygous translocation T6-9e stock obtained from the Maize Genetics Cooperation in 1961 (stock no. 57-413-2) by D.S. Robertson. To analyze the distribution of carotenoids in the maize kernel we extracted total carotenoids separately from endosperms and embryos. The carotenoids were separated and quantified by using high pressure liquid chromatography. The major carotenoids found in the kernels were zeaxanthin/lutein, a xanthophyll monoester, Y1 (Table 1). The types of carotenoids present in the endosperm of kernels homozygous for the dominant or recessive allele of Y1 were the same, however, they were found in significantly lower quantities in the endosperm of homozygous recessive kernels (Table 1).

Table 1. Distribution of carotenoids in maize kernels^a.
 

	Lutein/zeaxanthin	b-cryptoxanthin	b-carotene	a-carotene
	µg/g	µg/g	µg/g	µg/gb
Y1 Y1 embryo	16.54 ± 3.20	3.86 ± 1.14	2.07 ± 0.43	0.55 ± 0.24
y1 y1 embryo	12.65 ± 1.14	2.80 ± 0.80	2.72 ± 1.03	0.86 ± 0.29
	N.S.	N.S.	N.S.	N.S.c
Y1 Y1 Y1 endosperm	10.00 ± 0.59	0.54 ± 0.07	2.02 ± 0.07	0.33 ± 0.07
y1 y1 y1 endosperm	0.62 ± 0.32	0.23 ± 0.03	0.29 ± 0.06	0.08 ± 0.02
	p<0.001	p<0.001	p<0.001	p<0.01c

^aEach value represents the mean ± standard deviation of embryo or endosperm samples from three separate ears measured in triplicate, with the exception of the Y1 Y1 embryos, which were done in duplicate.
^bData are expressed as µg carotenoid per gram of wet tissue.
^cValues were compared by using an unpaired t test. Samples were not considered to be significantly different (N.S.) if the calculated p value was greater than 0.05.

The Y1 gene codes for phytoene synthase, the enzyme that converts two molecules of geranylgeranyl pyrophosphate to phytoene, the first C40 carotenoid. RNA blot hybridization analyses were performed to investigate the expression of the Y1 gene. Rehybridization of the RNA blots used in this study with a human actin hybridization probe indicated that all samples contained RNA. Densitometric analysis of the RNA blot hybridization autoradiographs allowed us to compare the amount of Y1 mRNA present in each tissue. Embryos isolated 30 days after pollination (DAP) from kernels which were homozygous for the dominant or recessive allele of Y1 both contain Y1 mRNA, however, the Y1 mRNA in the Y1 Y1 embryo was approximately 6 times more abundant. A Y1 mRNA transcript was detected in endosperm isolated from 30 DAP kernels which were homozygous for the dominant allele of Y1, however, no transcript was detected in 30 DAP endosperm which was homozygous for the recessive allele of Y1.

There was no significant difference in the amount of carotenoids detected in the embryo of kernels homozygous for either the dominant or recessive alleles of Y1. However, approximately 6 times more Y1 mRNA was detected in embryos that were homozygous for the dominant allele of Y1. There are several hypotheses that might account for this observation. One possibility is that the quantity of carotenoids in 30 DAP embryos is reflective of a peak amount of Y1 gene expression that occurred prior to 30 DAP. An analysis of the expression of the Y1 gene during the development of kernels may indicate if this is the case. Alternatively, if the biosynthetic steps subsequent to phytoene synthesis, but prior to the synthesis of the carotenoids analyzed in this study, are rate limiting, then phytoene may accumulate in the embryos of both genotypes. If this were true, then the lower level of Y1 mRNA present in the embryos that are homozygous for the recessive allele of Y1 might be sufficient to produce the level of colored carotenoids measured in this study. Analysis of phytoene and all subsequent carotenoids in the embryo of both genotypes should address this possibility.

Carotenoids were detected in 30 DAP endosperm of plants that were homozygous for the standard recessive allele of Y1, however, no Y1 mRNA was detected in this tissue. The inability to detect Y1 mRNA in this tissue may be due to the Y1 gene being expressed in this tissue at an earlier time or due to the expression of the Y1 gene below the level of sensitivity afforded by RNA blot hybridization analysis. Using a reverse transcriptase and polymerase chain reaction assay for expression should indicate if and when the recessive allele of Y1 is transcribed in the endosperm of plants which are homozygous for the recessive Y1 allele.

Alternatively, if maize has more than one phytoene synthase gene, the additional locus (loci) may be expressed in endosperm of plants that are homozygous for the recessive allele of Y1 and could thereby be partly responsible for the presence of carotenoids at the low levels found in this tissue. Additional phytoene synthase loci would also explain why no albino or viviparous alleles of Y1 have been described. DNA blot hybridization analysis using the Y1 gene as a hybridization probe and high stringency hybridization and wash conditions often exhibits DNA fragments in addition to those expected from restriction endonuclease map and sequence data of the cloned Y1 gene. These additional DNA fragments do not hybridize to the same extent as the Y1 sequences and might represent other loci that are members of a phytoene synthase gene family. Transcription of multiple phytoene synthase loci may not have been detected in this study if transcripts are present at a concentration below the level of detection afforded by RNA blot hybridization analyses or if the sequence of the non-Y1 phytoene synthase loci has diverged significantly from the Y1 sequence. 

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