Overexpression Of TsVP Improves Low Phosphate Tolerance In Maize And Comparative Analysis Of Low Phosphate Tolerance-associated MicroRNAs In Two Maize Genotypes

Phosphorous （P） is a macronutrient that is essential for plant growth, development, and reproduction. Despite the importance of P in agricultural production, most P in the soil is unavailable for plant because of the low availability of soluble phosphate （Pi）, the major form of P absorbed by plant roots. Therefore, Pi availability is usually a constraint on plant productivity in many natural and agricultural ecosystems Maize （Zea mays） is an important grain and forage crop worldwide. Pi availability is critical in the early developmental stages of maize and therefore has an important effect on production. However, maize is adversely affected by Pi deficiency in many areas where it is grown, particularly in the acid soils of tropical and subtropical regions and the calcareous soils of temperate regions. These soils account for more than half the area under maize cultivation. The use of Pi-rich fertilizer can improve crop yields that are limited by Pi deficiency but this practice is costly and dangerous to aquatic ecosystems because of the resulting eutrophication. Therefore, understanding of the mechanism behind Pi tolerance in maize and development of maize crops with improved low Pi tolerance is of significance to both agriculture and the environment.Overexpression of Thellungiella Halophila H+-pyrophosphatase gene TsVP improves low phosphate tolerance in maizeEngineering a crop with enhanced low phosphate tolerance by transgenic technique could be one way of alleviating agricultural losses due to phosphate deficiency. Many studies suggested that the overexpression of vacuolar H-pyrophosphatase （H-PPase） gene could enhance abiotic stress tolerance and improve root growth in many plants. As the primary organ involved in the efficient uptake of all the mineral elements, more robust root systems can facilitate Pi uptake, it is important for improving low Pi tolerance in plants To test whether the H-PPase could improve low Pi tolerance in maize, transgenic maize plants that overexpressed1helhingiella Halophila H-PPase Gene TsVP were tested for their performance under Pi deficit stress conditions.Maize seedlings were cultured in SP （sufficient phosphate,1,000μM KH2PO4） nutrient solution for20days, then divided into two groups and grown separately in SP and LP （low phosphate,5μM KH2PO4） nutrient solutions for an additional25days After25days of Pi deficit stress, all plants were adversely affected However, the transgenic plants displayed less growth retardation than the wild type, they showed significantly higher shoot biomass、root biomass and plant biomass than wild type （P P<0.05）, suggesting that transgenic plants had enhanced low phosphate tolerance Under both SP and LP conditions, the root dry weight and root dry weight to shoot dry weight ratio in transgenic plans were also significantly higher than in the wild type （P<0.05）. Moreover, transgenic plans had more axile roots, more lateral roots, longer total root length, larger root volume, larger root absorptive surface area and longer average lateral root length compared to the wild type under both SP and LP conditions. These data suggested that overexpression of TsVP in maize improved root growth significantly. There were no significant differences （P>0.05） in root soluble sugar content and root sugar content/shoot sugar content ratio between wild type and transgenic maize plants under both SP and LP conditions, suggesting that the distribution of soluble sugar in plant was not the reason for the enhanced root growth in transgenic plants The IAA content was higher in roots of transgenic plants than in roots of the wild type By contrast, the IAA content was lower in shoots of transgenic plants than in shoots of the wild type. But there was no difference in the total IAA content between the wild type and transgenic plants. Moreover, the expression levels of some genes involved in auxin transport were analyzed by real-time PCR, including ZmPINla, ZmPINlb and ZmAUX1. Transgenic maize plants showed a significantly higher （P<0.05） relative expression level of these genes compared to the wild type plants. These results suggested that overexpression of TsVP in maize might change the transport of1AA.1AA or NPA were added to wild type and transgenic plants for14days, after14days of treatment with IAA or NPA, there no significant differences （P>0.05） in root growth between wild type and transgenic plants anymore. These results suggested that IAA might be involved in the enhanced root growth in transgenic plants.Transgenic plants showed significantly lower （P<0.05） pH value of nutrient solution than wild type under Pi deficiency, which suggested that transgenic maize plants had a enhanced rhizosphere acidification ability compared to wild type. There were no significant differences （P>0.05） in organic acid secretion rate between wild type and transgenic plants, suggesting that organic acid secretion rate was not the reason for the enhanced rhizosphere acidification ability in transgenic plants. The activity of plasmalemma H+-ATPase （P-ATPase） was significantly higher （P<0.05） in transgenic plants than in wild type under both SP and LP conditions, suggesting that overexpression of TsVP in maize significantly increased the activity of P-ATPase, which was the reason for the enhanced rhizosphere acidification ability in transgenic plants. These results indicated that the enhanced root growth and increased rhizosphere acidification ability caused by higher （P<0.05） P-ATPase might play important role in improving low phosphate tolerance in transgenic maize plants.Transgenic maize plants had significantly higher （P<0.05） Imax values than the wild type plants, however, the Cmin and Km values in transgenic plants showed no significant differences （P>0.05） from the wild type plants under both of SP and LP conditions, which indicated that there was no difference in the affinity of Pi transporters for Pi between transgenic plants and wild type. Therefore, it could be concluded that the higher Imax values seen in transgenic plants were not due to the difference in the affinity of Pi transporters for Pi, might be due to the larger root systems in transgenic plants. Transgenic maize plants accumulated greater amount of P in shoots and roots than the wild type, this result was consistent with the higher Pi influx rate in transgenic plants.Whether under SP and under LP conditions, APase activity in the transgenic plants showed no significant difference （P>0.05） from the wild type, which suggested that the less growth retardation under Pi deficit stress and enhanced performance displayed by transgenic plants had no correlation with the APase activityIn addition to the enhanced low Pi tolerance compared to the wild type when cultured under hydroponic conditions, maize plants that overexpressed TsFP also showed improved performance compared to the wild type plants when grown in low Pi soil. The transgenic plants showed improved root growth and shoot growth in low Pi soil compared to the wild type. The photosynthetic rates and stomatal conductance value were smaller while the intercellular CO2concentration was higher in maize grown under LP conditions compared to maize grown under SP conditions, suggesting that the decrease in photosynthetic capacity caused by low phosphate stress was mainly due to non-stomatal factors. The photosynthetic rates and stomatal conductance value were higher in transgenic plants than in wild type plants while the intercellular CO2concentration was lower in transgenic plants than in wild type plants, suggesting that the decrease in photosynthetic capacity in wild type was mainly due to non-stomatal factors Possibly because of the higher photosynthetic capacity, transgenic plants produced higher grain yield per plant than wild type plantsIn summary, the results showed that the overexpression of TsVP in maize plants improved root growth and enhanced rhizosphere acidification and these phenotypes can help confer a higher degree of low phosphate tolerance to transgenic plant Most importantly, overexpression of TsVP in maize plants could increase grain yield per plant of maize under low Pi stress. This research indicated that the TsVP gene has the potential to be used for improving crop’s low phosphate tolerance and yields in areas where low Pi availability is a limiting factor for agricultural productivityIdentification and comparative analysis of low phosphate tolerance-associated microRNAs in two maize genotypesMicroRNAs （miRNAs） are known to play critical roles in plant responses to low Pi stress. The identification of low Pi tolerance-associated miRNAs can help in the selection and manipulation of high performing maize genotypes under low Pi-fertilizer conditions. Although numerous Pi starvation-responsive miRNAs have been identified in several plants, miRNAs associated with low Pi tolerance have not been identified. The comparison of miRNA expression profiles of maize genotypes differing in low Pi tolerance should provide useful information that will lead to the identification of low Pi tolerance-associated miRNAs and improve understanding of the mechanism behind low Pi tolerance in plants.We grew two maize genotypes （wild type, Qi319, and a low-Pi tolerant mutant,99038） under SP （sufficient phosphate,1,000μM KH2PO4） and LP （low phosphate,5μM KH2PO4） conditions for11days. As expected, under LP conditions, the two maize genotypes were significantly different with regards to low Pi tolerance. The growth of the99038plants was less affected by the lack of Pi. The99038plants accumulated significantly higher root biomass and plant biomass than the Qi319plants （P<0.05）. Moreover, under both of SP and LP conditions, the number of lateral roots, the lengths of the lateral root and the axile root, the total root length, and the root-shoot ratio increased significantly （P<0.05） in99038compared to the Qi319genotype.The maize roots were collected in order to construct small RNA libraries （Qi319SP,99038SP, Qi319LP, and99038LP）. The libraries were sequenced using Solexa technology. From the Qi319SP,99038SP, Qi319LP, and99038LP libraries, we identified193,205,208, and212known miRNAs belonging to25,25,26, and26miRNA families, respectively. We also identified46,59,50, and54novel miRNAs, respectively. The length of the predicted novel miRNA precursors varied from67to331nt, with an average of159nt. The length of the mature miRNAs ranged from20to23nt, the majority were21nt in length. The minimum free energy （MFE） values of precursors were in the range:-18.4to-128.3kcal mol-1, with an average of-60.59kcal mol-1. We were able to predict targets for26known maize miRNA families. These targets included transcription factors, such as SBP （miR156）, MYB （miR159, miR164, miR319）, ARF （miR.160）, NAC （miR164）, NFYA （miR169）, AP2-type transcription factors （miR172）, and GAMYB （miR319）. Other miRNA targets include genes involved in nutrient homeostasis, such as ammonium transporters （miR162）, ATP sulfurylase （miRNA395）, phosphate transporters （miR399）, Cu/Zn superoxide dismutase CSDs （miR398）, SPX domain proteins （miR827）, and genes implicated in plant development, such as HD-ZIP proteins （miR166） and laccases （miR397, miR528）, and other genes with diverse functions, such as phosphatase （miR393, miR319）, ascorbate oxidase （miR397）, blue copper protein （miR408）, and50S ribosomal protein （miR167） In many cases, some targets were identified as uncharacterized protein. The target genes for56novel miRNAs were successfully predicted. The novel miRNAs from maize target a variety of genes involved in diverse biological functions, including transcription factors, various enzymes. structural proteins, and transport proteins. Most miRNAs had multiple target sites, which suggested that these miRNAs have different functions. In most cases the targets were identified as uncharacterized protein However, for30novel miRNAs, we failed to discover any targets for them in maize As complete annotations for the sequence information in maize become more available, then more accurate target prediction and verification will be possible.In order to identify the differentially expressed miRNAs between Qi3l9and99038, the expression level of miRNAs between the two maize genotypes were compared under SP and LP conditions. Under SP conditions, members of four known miRNA families （miR160, miR164, miR.397, and miR528） and five novel miRNAs （mir20, mir₁29, mir₄5, mir₁72, and mir₂06） were differentially expressed between Qi319and99038. Among them, members of the miRI60family and three novel miRNAs （mir20, mir₁72, and mir₂06） were up-regulated, whereas the others were down-regulated in99038SP compared to Qi319SP Three novel miRNAs （mir₂0, mir₁72, and mir206） were only expressed in99038SP, whereas the other two （mir₄5and mir₁29） were only expressed in Qi3l9SP. Under LP conditions, the number of differently expressed miRNAs between the genotypes was greater than that under SP conditions. Overall, under LP conditions, members from six known miRNA families （miR1432, miR160, miR169, miR164, miR397, and miR528） and five novel miRNAs （mir₉9, mir₁00, mir45, mir₁72, and mir₂06） were differentially expressed between Qi319and99038. Members of four known miRNA families （miR160, miR164, miR397, and miR528） and three novel miRNAs （mir₄5, mir₁72, and mir206） were differently expressed between genotypes, regardless of the treatments. However, miR1432, miR169a-3p, miR169b-3p, and two novel miRNAs （mir₉9and mir₁00） were found to be differentially expressed between Qi319and99038under the LP conditions only and mir20and mir₁29were differentially expressed between Qi319and99038under the SP conditions only.In order to identify the Pi starvation responsive miRNAs, miRNA expression levels between low Pi stressed samples and their corresponding control samples for each genotype were compared. Ten known miRNA families and six novel miRNAs were significantly regulated by low Pi stress in both Qi319and99038. Most of Pi starvation responsive miRNAs were down-regulated by low Pi stress and only two known miRNA families （miR399and miR827） were up-regulated, which suggested that down-regulation of miRNAs appeared to be more important in low Pi response However, five novel miRNAs （mir₁29, mir₂0, mir₄5, mir₉9, and mir₁00） were regulated by low Pi stress in only Qi319and one （mir₂06） in only99038.Eight of the miRNAs that were differentially expressed between the genotypes and nine low Pi responsive miRNAs were selected for validation of their expression levels by real-time PCR. The expression analysis showed a good consistency between the results derived from the high-throughput sequencing and the real-time PCR results.In order to study the expression pattern of miRNAs at different stages of low Pi stress, real-time PCR was performed on the leaves and roots of Qi319and99038at different time points （1,3,7,11, and14days） after low Pi stress for six miRNAs （miR160, miR164, miR397, miR398, miR399, and novel miRNA, mir₂06）. The99038genotype showed significantly higher （P<0.01） expression levels of miR160and mir₂06and lower （P<0.01） expression levels of miR164and miR397in roots than Qi319at several time points （1,3,7,11, and14days） after low Pi stress. The expression profiles of some predicted target genes were analyzed by real-time PCR. These targets included auxin response factor gene ARh’2targeted by miR160, MYB transcription factor and NAC domain transcription factor gene NAC5targeted by miR.164and laccase gene LAC4targeted by miR397. The expression levels of these target genes were also significantly different （P<0.01） between two maize genotype roots at several time points （1,3,7,11, and14days） after low Pi stress These results suggested that the differentially expressed miRNAs between genotypes identified by the sequencing analysis at11days after low Pi stress, were also differently expressed between genotypes at other growth stages and caused different expression of some of their target genes between genotypes Many studies suggested that these target genes are involved in root development in plants or play important roles in the transcription regulation of Pi starvation responses in plants.In summary, we compared miRNA expression levels in a low Pi tolerant mutant and a wild type maize genotype under different Pi conditions by deep sequencing technology. This led to the identification of six known miRNA families and seven novel miRNAs that were differently expressed between genotypes By comparing the expression level of miRNAs in the low Pi stressed samples to the control samples, we also identified10known miRNA families and12novel miRNAs that were responsive to Pi starvation in maize. Subsequent target gene prediction and expression analysis suggested that differently expressed miRNAs might play important roles in root development and transcription regulation of Pi starvation responses, which might contribute to different levels of low Pi tolerance in different maize genotypes. This study identified some miRNAs might help in improving low Pi tolerance in maize, leading to better understanding of the mechanism behind low Pi tolerance in maize.