| Sucrose is the main transport form of carbohydrate in higher plants. The transport direction and partioning efficiency of sucrose influence the plant growth and even are an important factor limiting grain yield. Sucrose transporters (SUCs or SUTs) in plasmalemma as important carriers are primarily responsible for exporting sucrose from source cells, loading and unloading of phloem, and importing sucrose to sink cells, thereby SUCs impact on transport direction, distributing efficiency and distribution of sucrose; and the expression of SUCs are greatly affected by various factors such as endogenous hormone, biotic stress and physicochemical stress. Thus, the further study on the relationship among expression of SUCs gene, environmental factor and hormone is the key to elucidate regulatory mechanism of photoassimilate transport and partioning under environmental stress.Sucrose transporters are encoded by a multi-gene family, which is composed of nine AtSUCs in model plant Arabidopsis. They have been identified and can be classified into three distinct subfamilies:SUT1-clade, SUT2-clade and SUT4-clade. The results of gene knockout indicate that complementary effects are detected between some family members and some knockout mutants lead to phenotype variation only under some certain conditions, suggesting that there are differences in the regulatory mechanism of SUCs expression, thus the gene expression of different members also varies in response to environmental stress. ABA is one of important plant hormones and a critical hormone in response to stress, also called stress hormone. It plays a crucial role in plant stress tolerance, including photoassimilate transport and partioning under environmental stress. However, the further study on relationship between SUCs and ABA has not been reported nearly to date.In this research,Arabidopsis was used as material, through microarray data and real-time quantitative reverse transcription PCR(qRT-PCR) analyses, we found that the high affinity/low capacity AtSUC2gene and low affinity/high capacity AtSUC4gene were key genes in response to salt, osmotic, drought, low temperature and exogenous ABA in Arabidopsis. The physicochemical stresses and ABA treatments induced the changes of growth, sucrose contents, ABA responsive genes and ABF-downstream and upstream genes in AtSUC2and AtSUC4homozygous mutants. Furthermore, we further discussed the effect and mechanism of AtSUC2and AtSUC4in response to physicochemical stress. We have also explored interaction of gene expression among AtSUC2, AtSUC4and other AtSUCs, and possible mechanism that ABA is involved in regulating the expression of AtSUCs. The main results are as follows: 1. By the analysis of microarray data, we found that the expression of AtSUC2and AtSUC4gene almost obviously increased in response to salt, osmotic, drought, low temperature, revealing that AtSUC2and AtSUC4may be positive regulators under physicochemical stresses. The expression of AtSUC1and AtSUC5almost markedly decreased in response to4stresses, indicating that AtSUC1and AtSUC5are possible negative regulators under physicochemical stresses. In addition, the expression of AtSUC3was only induced by the stresses at specific treatment times, whereas hardly significant changes in expression were seen for AtSUC6, AtSUC7, AtSUC8and AtSUC9for any stress treatments, suggesting that these genes might have a minor role in plant stress tolerance. Thus, AtSUC2and AtSUC4may be the key genes that positively respond to physicochemical stress.2. We further investigated the expression of AtSUC2and AtSUC4by qRT-PCR in WT under salt, osmotic, drought and cold stresses, and found that the expression of two genes enhanced significantly in WT under physicochemical stresses. With the treatment time increasing, the expression of two genes increased first and reached the maximum, and then decreased; and all expression under physicochemical stresses were higher than control. Thses results indicated that4physicochemical stresses induced the high expression of AtSUC2and AtSUC4.3. The homozygous mutants of Atsuc2-1, Atsuc2-3, Atsuc3, Atsuc4-1and Atsuc4-2were identified precisely by three primers PCR and reverse transcription PCR (RT-PCR), and5mutants were used as experimental materials of our subsequent investigations.4. In the control condition, there was obviously different performance between mutants and WT, for example, lower germination, fewer leaves, shorter roots, and smaller leaf area. Under salt, osmotic and cold stresses, the difference of germination between WT and mutants was more obvious. With the increase of treatment time, the difference of germination reduced gradually. Physicochemical stresses only delayed but not inhibited the germination of seed, thus3stresses had no effect on the final germination percentage of seeds. During seedling stage, these phenotypic differences became more significant with the increase of stress strength, suggesting that the disruption of the AtSUC2and AtSUC4genes led to hypersensitivity to physicochemical stresses.5. In the control condition, we found sucrose contents in the mutant shoots closed to each other and were higher than in WT shoots, whereas sucrose contents for the mutant roots were similar and lower than for WT roots. Exposure to salt, osmotic and cold stresses resulted much higher sucrose contents in shoots and roots of WT and mutants compared with sucrose contents in the control condition, much higher sucrose contents in mutant shoots than in WT shoots, and much lower sucrose contents in mutant roots than in WT roots, and the difference was statistically significant. The results indicate that physicochemical stresses inhibit the transport and partioning of sucrose from source leaves to roots, and AtSUC2and AtSUC4play a critical role in the process.6. By investigating AtSUC2and AtSUC4expression in Atsuc3under normal and physicochemical stress conditions, we found that disruption of AtSUC3weakened the increase in AtSUC2and AtSUC4expression induced by salt, osmotic, drought and cold stresses, indicating that AtSUC3may be involved in modulating the expression of AtSUC2and AtSUC4under physicochemical stress.7. We investigated the expression of AtSUC2and AtSUC4by microarray data and qRT-PCR in WT under exogenous ABA treatment, the results showed that exogenous ABA induced the high expression of AtSUC2and AtSUC4. We found that loss-of-function mutants of AtSUC2and AtSUC4showed hypersensitivity to exogenous ABA during seed germination and seedling growth, such as lower germination, fewer leaves, shorter roots, and smaller leaf area. Exogenous ABA treatments induced higher sucrose content in shoots and lower sucrose content in roots of these mutants compared with WT, revealing that the disruption of the AtSUC2and AtSUC4inhibit the transport of sucrose from source leaves to roots. In addition, disruption of the AtSUC2and AtSUC4also inhibited the ABA-induced expression of many stressES and ABA responsive genes, especially ABFs and ABF-downstream and upstream genes. The results show that AtSUC2and AtSUC4are involved in ABA signal transduction.In sum, AtSUC2and AtSUC4are key genes in AtSUC gene family in response to salt, osmotic, drought, cold stresses and exogenous ABA. The loss-of-function mutants of AtSUC2and AtSUC4resulted in accumulation of sucrose in source leaves, decreasing of sucrose contents in roots, the delay of seed germination and inhibition of seeding growth. Furthermore, disruption of the AtSUC2and AtSUC4also inhibited the ABA responsive genes and ABF-downstream and upstream genes. These findings confirmed that AtSUC2and AtSUC4are important regulators in plant physicochemical stress tolerance and operate an ABA-dependent signalling pathway. In addition, disruption of AtSUC3weakened the increase in AtSUC2and AtSUC4expression induced by physicochemical stresses and exogenous ABA. Presumably, AtSUC3is a sucrose sensor, sensing the changes of sucrose concentration in cell and regulating the activity of other AtSUCs; the results in this research also support this opinion. |
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