| The screening, breeding and exploiting of cultivars that accumulate low heavy metals in edible part has been a hot point to cope with the heavy metal pollution in agriculture soil. It has been reported that plants belonging to cruciferae genus Brassica are high heavy metal accumulator and could be easily polluted by heavy metals such as Cd and Pb. In order to minimize the entry of Cd into the food chain, the present study investigated the Cd accumulation of Chinese flowering cabbage （Brassica parachinensis）, a member of the genus Brassica, for selection of low Cd cultivars. Several tapical cultivars were obtained, and the stability of Cd accumulative characteristics was investigated in different soils and different harvest times. Then, the effets of agricultural approaches such as culture mixture and P application on the Cd accumulation of the typical cultivars were evaluated. The subcellular distribution and chemical speciation of Cd in the typical cultivars were investigated with methods of centrifugation technique and successive extraction, to explain the main biochemical mechanisms of cultivar variation of Cd accumulation and the inhibition effect of P application on Cd accumulation. We also analyzed the single mucleotide polymorphism （SNP） of 17 Chinese flowering cabbage cultivars and investigated the feasibility of identifying low Cd cultivars with molecular biomarkers. This study provided theoretical basis to specific the biochemical and molecular michanisms of the low Cd accumulative characteristic of Chinese flowering cabbage. It also provided the scientifical evidence for the combination of agricultural approaches with Cd-PSC （pollution safe cultivar） stratigy to lower the risk of Cd pollution in Chinese flowering cabbage and ensure the safe production of Chinese flowering cabbage in practical agricultural actions. The main results were as below:1. A pot experiment was conducted to investigate Cd accumulation of 31 Chinese flowering cabbage cultivars under CK (0.114 mg kg-1), LCd (0.667 mg kg-1) and HCd (1.127 mg kg-1) treatments. The shoot Cd concentrations of all cultivars exceeded the maximum limit of CAC for Cd (0.2 mg kg-1,FW) under the LCd treatment, and the Cd concentration of CLW was even 10 times higher of this limit under HCd treatment. These results indicated Chinese flowering cabbage could readily take up Cd from polluted soils. The variance analysis showed that the variety effect on the shoot Cd concentration of Chinse flowring cabbage was prominent at level of p < 0.01. The shoot Cd concentrations of the tested cultivars under CK, LCd and HCd treatments were 0.033-0.162 mg kg-1, 0.283-1.743 mg kg-1and 0.434-2.276 mg kg-1. The maximum differences of Cd concentration between the cultivars under the same treatment were 4, 5.2 and 5.6 folds, enough for the seletion of low Cd cultivar. The correlation of shoot Cd concentrations between each treatment were significant at p < 0.01 level, showing the stabllity of Cd accumulation of Chinese flowering cabbage. Six cultivars, SJ19, 49CX, XG49, CH4, LB70 and YL80 were selected as typical low Cd cultivars, and CLW was typical high Cd cultivar.2. The responses of the typical cultivars to the Cd and Pb compound pollution were investigated using a pot experiment. The variety effect on the shoot Cd concentration of Chinese flowering cabbage were significant at p < 0.01 level under all treatments. The average Cd concentrations of six low Cd cultivars were 0.079 - 0.097 mg kg-1, while that for the high Cd cultivar was 0.242 mg kg-1. Shoot Cd concentraions of 6 low cultivars were always significantly lower （p < 0.01） than those of the high Cd cultivar, CLW. Shoot Pb concentration of 7 tested cultivars were 0.145 - 0.200 mg kg-1, in the decreasing order of CLW > YL80 > XG49 > SJ19 > 49CX > LB70 > CH4. The high Cd cultivar accumulated the highest Pb in shoots than other cultivars under most treatments. The variance analysis showed the interaction between Cd and Pb was significant （p < 0.01）. The soil Pb (51.02 - 91.25 mg kg-1) obviously inhibited the Cd accumulation of Chinese flowering cabbage. According to the definition of CAC and PSC, all the low Cd cultivars could be treated as Cd+Pb-PSCs, and the high Cd cultivar was non-Cd+Pb-PSCs under the soil condition in this study (Cd: 0.111 - 0.383 mg kg-1; Pb: 23.50 - 91.25 mg kg-1).3. The present study investigated the effects of different soils and harvest times on the typical cultivars of Chinese flowering cabbage. The shoot Cd concentrations of six typical cultivars in soil A and soil B were 0.026 - 0.080 mg kg-1 and 0.452 - 1.349 mg kg-1, respectively. Both the shoot and root Cd concentraions of all cultivars in soil A were significantly （p < 0.01） lower than those in soil B, with the maximum differences being 20 and 13 folds, respectively. It might have relationship with the extremely higher soil available P content in soil A as compared with soil B. The shoot Cd concentrations and transfer ratios of high Cd cultivars, CLW and CX4, were always significantly higher than those of low Cd cultivars, SJ19, CH4, LB70 and YL80, indicating the Cd accumulative characteristics of typical cultivars were stable under different soils. The average shoot Cd concentrations of all tested cultivars for harvest time T1 （25th day）, T2 （40th day） and T3 （60th day） were 0.734 mg kg-1, 0.569 mg kg-1 and 0.527 mg kg-1, respectively. The shoot Cd concentrations of all tested cultivars were highest in the initial growth stage （T1）, decreasing as time goes, with the most obvious decrease from T1 to T2. The average Cd amount absorbed by the tested cultivars each day for the T1, T2 and T3 were 0.46μg pot-1 day-1, 0.87μg pot-1 day-1and 0.01μg pot-1 day-1, respectively, showing the fastest absorption speed of Cd in the middle stage （T2） of growth, being 1.9 folds of initial stage （T1）. The shoot Cd concentrations of low Cd cultivars, SJ19 and CH4, were always significantly lower than those of high Cd cultivars, CLW and CX4. The amount of total Cd in shoots of low Cd cultivars were only 1/3 - 1/2 of those of high Cd cultivars, showing that the harvest time would not change the Cd accumulative characteristics of typical cultivars.4. Taking the Cd-PSCs （SJ19 and CH4） and non-Cd-PSCs （CLW and CX4） as objects for study, the cultivar mixture on the Cd accumulation of typical Chinese flowering cabbage cultivars was investigated. The shoot Cd concentrations of CLW, SJ19, CH4 and CX4 were 0.485 - 0.551、0.274 - 0.522、0.233 - 0.423 and 0.643 - 0.775 mg kg-1, respectively. The shoot Cd concentrations of SJ19 and CH4 under cultivar mixtures were significantly （p < 0.05） lower than those of CLW and CX4, except that the Cd concentrations of SJ19 and CH4 were a little high when co-cropped with CX4. There was no significant effect of cultivar mixture on shoot Cd concentrations when the low Cd cultivars co-cropped with high Cd cultivar“CLW”, but the shoot Cd concentrations of the low Cd cultivars increased significantly （p < 0.05） when they were co-cropped with another high Cd cultivar“CX4”. When the low Cd cultivar“SJ19”co-cropped with the other low Cd cultivar“CH4”, the shoot Cd concentrations of both culticars significantly （p < 0.05） decreased, with decreses of 24.6%-26.2% and 25.8%-28.7%, respectively. The results showed that rhizosphere plays an important role in the Cd accumulation of Chinese flowering cabbage, and the selection of proper cultivar mixture will effectively decrease the risk of Cd pollution of Chines flowering cabbage.5. This study investigated the soil Cd fractions and the Cd accumulation of three Cd-PSCs and two non-Cd-PSCs under different soil P levels. The concentrations and proportions of exchangeable Cd in the CK soil and rhizosphere soils of CH4 and CX4 decreased with the increasing soil P level, but those of Fe-Mn oxide Cd showed the opposite trend. It assumed that P applying decreased the bioavailability of soil Cd, and thus reduced the total amount of Cd absorbed by Chinese flowering cabbage. The shoot Cd concentrations of all tested cultivars decreased with the increasing of soil P levels. Compared with P1 treatment, the shoot Cd concentrations of CLW, CX4, SJ19, CH4 and LB70 under P4 treatment decreased by 64%, 68%, 50%, 63% and 63%, respectively, and the translocation factors of Cd decreased by 33.6%, 35.4%, 23.9%, 48.3% and 28.0%, respectively. The increase of soil P level not only decreased the shoot Cd concentration of Chinese flowering cabbage, but also effectively inhibited the translocation of Cd from root to shoot. The non-Cd-PSCs always showed higher Cd shoot concentrations than Cd-PSCs, indicating there was no effect of soil P on the Cd accumulative charateristics of the typical cultivars.6. The subcellular distribution and chemical speciation of Cd in LB70 and CX4 under different soil P levels were investigated with the methods of centrifugation technique and successive extraction. The results showed that the proportions of Cd bound to the cell wall fraction were obviously higher in LB70 than in CX4, but the proportions of Cd in the soluble fraction and organelle fraction were always higher in CX4 than in LB70. Compared to CX4, LB70 had lower proportions of Cd in inorganic form and water-soluble from, but higher proportions of Cd in proteins / pectates integrated form, implied that the Cd accumulation in LB70 is associated with the low mobility of Cd in vivo. The increase of soil P level obviously improved the proportions of Cd bound to cell wall fraction and the proportions of Cd in water-soluble form and insoluble Cd-phosphate complexes, but decreased the proportions of Cd in inorganic form. The P application decreased the mobility of Cd in vivo and inhibited the translocation of Cd from roots to shoots through the courses of enhancing the bouding of Cd to cell wall fraction and decreasing proportions of Cd in inorganic form.7. This study investigated the single mucleotide polymorphism of three membrane protein genes which were reported to be associated with Cd accumulation, Pcr1, HMA4 and NRAMP3, in 17 cultivars which were low, middle or high Cd accumulators, respectively. Abundant SNPs were obtained through sequence analysis. The frequencies of SNP in Pcr1, HMA4 and NRAMP3 were 1 SNP/34.4 bp, 1 SNP/50.8 bp and 1 SNP/34.7 bp, respectively. The frequency of transition was 2-10 folds of transversion, indicating the preference of the base substitution. The frequencies of Indel in Pcr1 and NRAMP3 were 1 Indel/218 bp and 1 Indel/84.9 bp, no Indel was found in HMA4, showing the HMA4 was more conservative than the other two genes. According to the nucleotide polymorphism, the Pcr1, HMA4 and NRAMP3 formed 8, 10 and 10 haplotypes, respectively. The haplotype 8 of Pcr1 included 5 low Cd cultivars, therefore, this haplotype was consistent with low Cd accumulation and could be used as a molecular marker for the assistant selection of low Cd cultivars of Chinese flowering cabbage. The cluster analysis showed that the sequences were similar among the low Cd cultivars which may have close genetic relationship. |
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