Quantitation of 18kDa HSP mRNA accumulation in metal-ion insulted maize seedling radicles and plumules
--Bouchard, RA, Yang, Z, Walden, DB

In a study employing in situ techniques (Yang and Walden, MNL 71:55, 1997), we previously reported that maize root tips insulted with the heavy metals Cd and Zn accumulate mRNA for 18kDa HSPs in the epidermal and cortex regions of the root tip. These tissues are distinct from the active meristems and immature vascular bundles where these messages accumulate during heat shock induction (Greyson et al., 1996. Devel. Genet. 18:244-253). We now report preliminary results of quantitative RNA-Dot experiments in which RNA from metal-ion insulted radicles and plumules was screened with a general ORF probe for 18kDa HSP mRNAs and also with gene-specific probes for three members of the 18kDa HSP gene family.

Seedling growth, heat shock (HS), and heavy metal-ion insults were performed exactly as reported in the note cited above, while RNA extraction, filter-binding, preparation of labeled DNA probes, hybridization conditions, scanning, and quantitation were as described in Bouchard et al. (Maydica 38:135-144, 1993). The probes were Mhsp18-9-2, a subclone containing the ORF of clone Mhsp 18-9 (map designation uwo11), which is a common probe for mRNAs from all members of the maize shsp gene family, and subclones Mhsp18-9-3, Mhsp18-1-1, and Mhsp18-3-3, which are gene-specific 3'-UTR regions for the three shsp family members with map designations uwo11, uwo9, and uwo10 repsectively. The results, normalized to the signals seen in the untreated control for each probe set to 100%, are shown in Tables 1 and 2.

Table 1. Radicles.
 

Treatment	C	HS	H2O	Zn	Cd	K	Na	Cu
Probe
18-9-2 (ORF)	100	2209	154	880	984	138	126	381
18-9-3 (uwo11)	100	11967	100	3257	4853	100	100	893
18-1-1 (uwo9)	100	14150	100	1285	900	395	395	615
18-3-3 (uwo10)	100	2302	269	201	166	137	106	111

Table 2. Plumules.
 

Treatment	C	HS	H2O	Zn	Cd	K	Na	Cu
Probe
18-9-2 (ORF)	100	1850	98	421	658	75	96	138
18-9-3 (uwo11)	100	18210	100	1550	4865	100	100	250
18-1-1 (uwo9)	100	11475	100	1905	1325	100	100	510
18-3-3 (uwo10)	100	1301	94	102	135	112	161	87

These results, though still preliminary, appear both to confirm and extend our earlier in situ study in which the ORF probe alone was used. Induction of overall 18kDa mRNA accumulation due to insults to radicles with the divalent heavy metal-ions Zn and Cd relative to both controls and monovalent ions is evident with the ORF probe, just as seen in the in situ experiments, and a similar pattern is observed in RNA from plumules. In addition, there appears to be a more modest but consistent induction of mRNA accumulation with Cu. However, some interesting differences emerge when the results seen with the specific gene probes are compared with the 18 kDa common ORF probe and with one another. The overall level of 18 kDa mRNA accumulation under heavy metal-ion insult observed with the ORF probe is highest in the presence of Cd. This is also the case for the RNA of 18-9; indeed, the relative level of induction appears even higher. In the case of 18-1, however, the level induced by Zn seems to be higher than Cd in both radicles and plumules. The most intriguing results are seen for the RNA of 18-3, which appears to show little or no consistent induction by the divalent heavy metal-ion insults when compared with either the control and H2O treatments or the monovalent heavy metal-ion insults.

The observations reported here suggest that the response to heavy metal-ion insult may exhibit specific differences among various members of the 18 kDa HSP gene family. This is consistent with observations we have previously reported for the developmentally modulated expression of these genes in the growing anthers and spikelets of maize (Bouchard et al., Maydica 38: 135-144, 1993). Interestingly, 18-3 shows no induction either from developmental cues or heavy metal-ion insults. This suggests that while the products of some members of the 18 kDa HSP gene family function in both heat stress and other contexts, the activities of others may be confined to heat stress conditions.

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