| Phosphate deficiency in soil and natural resources will be a potential agriculture crisis in the future. Low phosphate concentration is frequently a supply constraint in maize yields and increased the cost for production. At the same time, application of phosphate (Pi) fertilizer in large quantities produces the water pollution by organism in aquatic systems and degradation of the environment. It has been one of hotspots in plant biology to solve the above problems by exploring the potential ability of phosphate absorption of crops. Root postembryonic growth and development are critical for Pi uptake since for the poor mobility of soil phosphate, and root system could regulate the whole plant growth include the leaf aging, photosynthesis and stomata opening by synthesizing plant hormone such as cytokinins and abscisic acid. Maize is an important crop with unique features root system compared to the dicotyledonous model root system from Arabidopsis, and maybe the regulation mechanism was different. It is important to explore the development mechanisms of maize root in low phosphate environment for the improvement of the Pi efficiency by breeding.In this study, the gene expression profiles of root segments of inbred Q319 and 99038 (from a cell mutant) cultured in nutrient solution with different phosphate concentrations were examined using an Arizona Maize 47K Oligonucleotide Array. The expression pattern of transcription factor ZmPTF1 that was induced by low phosphate stress was determined, and the effects of ZmPTF1 expression levels on the maize root morphology and the function of ZmPTF1was carefully examined by transgenic strategy. Four auxin influx transporters genes and 13 auxin efflux transporters genes were cloned and the expression patterns of these genes and their promoter sequences were analyzed. Using Agrobacterium-mediated maize shoot transformation, the sense and antisense Zea mays auxin transporter protein 3 (ZmAUX1), ZmPIN1a and ZmPIN1b were introduced into inbred line DH4866. The morphology, development, yield, root architecture and their response to low phosphate stress of the transgenic homozygous lines were determined. The ZmPTFl and ZmPIN1a overexpressing lines which showed high prospects in maize breeding were obtained.The main results of this work are follows:Comparative transcriptome analysis of the inbred line Q319 and 99038 rootsIn this study, the gene expression profiles of root segments of inbred Q319 and 99038 were examined using an Arizona Maize 47K Oligonucleotide Array, which representing more than 30,000 identifiable unique maize genes. The samples were the root segments of the plants cultured in nutrient solution. And one kind of the segments were about 0.5cm of seminal root tips, and another were 1.0cm segment of seminal roots from 0.5cm to 1.5cm behind the root tip in which lateral root will take place. The genes of ratio≥1.5 or ratio<0.66 (99038/Q319) and p value less than 0.1 by t-test in the three independent biological repeats were defined as differentially expressed genes between the two genotypes. Before low phosphate treated, there are 470 up-regulated (99038/Q319) and 542 genes down-regulated in the root tips in 99038 compared with Q319, whereas in the segments where lateral root take place,621 genes were up-regulated and 572 genes were down-regulated. After 2d low phosphate culture, compare with Q319 grown in the same solution, there were 343 genes up-regulated in the root tips of 99038 and 245 genes down-regulated, while 272 genes up-regulated and 270 genes down-regulated in the segments where lateral root take place. After 8d low phosphate culture, in the root tip of 99038 there were 984 genes up-regulated and 812 genes down-regulated compare with the Q319, with 601 genes up-regulated and 457 genes down-regulated in the segments where lateral root take place. The catalog of GO indicated that Q319 and 99038 have their different low phosphate response profiles in the some point of treatment, also in the two segments. Among these genes, more than 50% have no hit in the database, others were belong to metabolism, cell signaling, transcription and cell proliferation and so on. The results indicated that it was a complex process of maize root response to low phosphate stress, and the intricate regulations and adaptation were existed.Before low phosphate treatment, genes involved in ethylene synthesis and signaling were differentially expressed in the two lines. 1-aminocyclopropane-1-carboxylic acid(ACC) oxidase (TM00025945、TM00042027) and acc synthase (TM00018872) which coding for the two enzymes in the synthesis of ethylene had lower expression in the segments where lateral root take place of 99038, but higher in the root tips. These suggested that the synthesis of ethylene via the adenosylmethionine may be different in the root segments of the two lines. The membrane binding protein EIN2 (ethylene insensitive 2, TM00057348) and the ethylene responsive transcription factor (ethylene-responsive factor-like protein 1, ERF1,TM00030445)and ethylene- responsive small GTP-binding protein (TM000 14304) which involved in the ethylene signaling showed a lower expression in the segments where lateral root take place of 99038 compare to Q319. The expression of ethylene responsive element binding factor 3 (TM00018574 and TM00042351) were high in the 99038 root tips. These indicated that ethylene might participate in the formation of more robust root system of 99038.Auxin was one of the key hormones in plant root development and the low phosphate response process. Auxin polar transport was essential to the local auxin accumulation, auxin signaling imitation and auxin regulated biological process. AUX1-like permease (TM00029483), which coding for an auxin influx transporter had a higher expression in both the root tip and segments where lateral root take place of line 99038, this might promote auxin to the root, and was a factor to form the root with more lateral roots of 99038. TM00003447, which represents a phosphatase 2A regulate subunit, had a lower expression in the segments where lateral root take place, the protein phosphatase 2A (PP2A) and PID (protein serine/threonine (Ser/Thr) kinase PINOID) act antagonistically on phosphorylation state of their central hydrophilic loop, hence mediating PIN apical-basal polar targeting. PGP1(TM00020888), which coding for another class of auxin efflux transporter was differentially expressed in the two lines. These differentially expressed auxin polar transporters and their regulation proteins could result in a different local auxin gradient in the root of 99038. TM00047995 which coding for IAA-Ala hydrolase role in the free IAA release from IAA-Ala, this gene was high expressed in the segments where lateral root take place of 99038, while had a lower expressed in the root tips. Tiyptophan synthase (TM00016868)、putative tyrosine/dopa decarboxylase (TM00005060) indole-3-glycerol phosphate lyase (TM00005958) and cinnamic acid 4-hydroxylase (YUCCA, TM00025513) were key enzymes participated in the biological synthesis of IAA. These differentially expressed auxin metabolism related genes may confer to the differential local auxin gradient in root of the two lines. Auxin binding protein1 (ABP1,TM00041857), auxin-induced in root cultures protein 12 (TM00055925) and GH3 family gene (TM00048104) and other auxin response genes and transcription factors showed different expression patterns in the two lines. The different levels of auxin synthesis, metabolism, transport, accumulation, signaling and the auxin regulated development process may be one of the main factors resulted in the differential root system of 99038 and Q319.The signaling of auxin and ethylene participate the acclimation of maize plants to the low phosphate stress. On the 2d and 8d low phosphate treatment, auxin polar transport and the regulation genes in root had differently expressed to the low phosphate stress between the two lines. It was possible that the auxin polar transport in the root was a key link in the responses to low phosphate stress and resulted in modification of the root morphology, this meant the low phosphate signaling regulated the root architecture by regulating the polar auxin transport and signaling. Compared with plants in SP solution, MAP3K、MAPK4、MAPK5、MAPK6 genes had an relative lower levels in the root tips of 99038, MAPK system usually function in coordination with ethylene signaling pathway, maybe the system participate the low phosphate response and regulate many aspects of plant to low phosphate stress. And the EIN3, ERF1 in the ethylene signaling pathway and Gibberellin-regulated protein 2 might directly involve in the lateral root occurrence and the acclimation of root to low phosphate environment. It was concluded that the different auxin and ethylene signaling between two genotypes was an important factor for their differences in low phosphate circumstance.Several transcription factors and signaling transduction genes, such as 14-3-3, ABP1, AUX1, RHD3, ROP6, SGR2 and BRIl1were differentially expressed between the two lines or in their response to low phosphate stress. This was inferred that the genes participated in the root morphology and low phosphate acclimation and could be the targets of root breeding and the breeding to tolerate low phosphate stress in maize.Overexpression of transcription factor ZmPTF1 improves low phosphate tolerance of maize by regulating carbon metabolism and root growthA bHLH (basic helix-loop-helix domain) transcription factor involved in tolerance to Pi starvation was cloned from Zea mays with an RT-PCR coupled RACE approach and named ZmPTFl in our previous work. ZmPTF1 encoded a putative protein of 481 amino acids that had identity with OsPTFl in basic region. Real-time RT-PCR revealed that ZmPTFl was quickly and significantly up-regulated in the root under phosphate starvation conditions. Overexpression of ZmPTF1 in maize improved root development in different mediums and the roots of the sense lines were significantly development than WT. When cultured in low phosphate soil, the sense plants developed more tassel branches and larger kernels and were less affected by low phosphate stress. Some low phosphate response genes have higher expression levels in the overexpression lines, such as drought induced RNase, vacuolar H+ pyrophosphatase (H+-PPase), PEP carboxykinase. Compared with wild type, overexpressing ZmPTF1 altered the concentrations of soluble sugars in transgenic plants, in which soluble sugars (Glu+Fru+Suc) levels were lower in the leaves and higher in the roots. Overexpression of ZmPTF1 enhanced the expression of fructose-1,6-bisphosphatase and sucrose phosphate synthasel participated in sucrose synthesis in the leaves but decreased them in the root, and reduced the expression of genes involved in sucrose catabolism in the roots. The modifications on the physiology and root morphology of the plants enhanced low phosphate tolerance and increased the yield under low phosphate conditions. This research provides a useful gene for transgenic breeding of maize that is tolerant to phosphate deficiency and is helpful for exploring the relationship between sugar signaling and phosphate concentrations in cells. These transgene lines had confirmed by experts in maize breeding and to be used to breed the variety with trait of tolerating low phosphate stress.Identification and expression analysis of AUX1/LAX family and PIN-formed family genes in maizeAuxin polar transport was critical for formation and maintaining of auxin concentration gradient around to the meristem, and this different auxin gradient initiate and regulates many aspects of plant growth and development. The polar auxin transport is mediated by three classes of proteins AUX1/LAX family, PIN-formed family and ABCB subfamily proteins. There are 4 auxin influx transporters and 8 PIN auxin efflux proteins in model plant Arabidopsis, whereas in monocots crop rice there are 4 auxin influx transporters and 12 PIN auxin efflux proteins respectively. Four putative auxin influx transporters and 13 putative PIN auxin efflux proteins were identified from the maize genome by a database search and 10 genes were cloned by RT-PCR. Phytogenetic analysis of auxin influx transporters indicated that the previously reported ZmAUX1 had the highest similarity to Arabidopsis auxin transporter 2 and 3, to the Os auxin transporter protein 3 in rice and renamed the gene Zm auxin transporter protein 3. The 13 auxin efflux transporters genes cloned by us were categorized into branches with Arabidopsis PIN genes and rice. In the PIN1 subfamily, there are four PIN1-like genes in rice and maize, and we named the four PIN1 genes in maize ZmPIN1a, ZmPIN1b, ZmPIN1c and ZmPIN1d sequentially. The mRNA sequences of ZmPIN1b and ZmPIN1c were derived from a identical genomic DNA sequence, we presumed the two mRNA came from one Primary transcript by the alternative splicing. ZmPIN1b and ZmPIN1c have a high similarity to OsPIN1a, while ZmPIN1a was the homologous gene of OsPIN1c, and ZmPIN1d was the homologous gene of OsPIN1b and OsPIN1d. There are two homologous genes of AtPIN3 in maize. There are five homologous genes of AtPIN5 in maize, they locate on the 3,4,8,2,1 chromosome respectively. A homologous gene of AtPINS and a homologous gene of OsPIN9 were found in maize and named them ZmPIN8 and ZmPIN9. The homologous genes of AtPIN2, AtPIN4, AtPIN6 and AtPIN7 did not exist in maize, and also none in rice of OsPIN4,OsPIN6 and OsPIN7.Promoter analysis of the genes pointed out that Zm auxin transporter protein 3 was more possible involved in drought stress, whereas Zm auxin transporter protein 2 and Zm auxin transporter protein 4 were mainly function in injury and wounding defense. In the PIN1 family, the response to high and low temperature were mainly taken on by ZmPIN1b/c, whereas the ZmPIN1a was function in drought and ABA response and regulated by endogenous or exogenous ABA, GA and ethylene signaling. ZmPIN3b might be the target genes of MYBHvl for there were 4 MYBHv1 binding sites in the promoter. These genes could be regulated by GA, zeatin and auxin.RT-PCR analysis showed that ZmAUX1 was highly expressed in root and shoot in the 4 days germinated seedlings, especially in the shoot, the expression level in the shoot was more than 5 fold of root. These suggested that ZmAUXl played an important role in the auxin transport from shoot to the root. Auxin influx transporter ZmPIN1b, ZmPIN1a have a relative higher expression to other genes, whereas the expression of ZmPIN3a、ZmPIN3b、ZmPIN5a、ZmPIN5b、ZmPIN5c、ZmPIN8、ZmPIN9 were very low in the 5-leaf stage. The high expression of these genes in the seed germination and organogenesis indicated that the participated the early morphogenesis of plant. They showed different expression patterns in the root tip, the segments where lateral root take place and the leaves of 5-leaf stage maize plant, and the response to low phosphate stress were diverse to the organs. In the segments where lateral root take place, ZmAUX1、ZmPIN1a、ZmPIN1b were up-regulated and ZmPIN1c was down-regulated by low phosphate stress.In the root tip and the leaf, ZmPIN1a was up-regulated by low phosphate stress, whereas ZmAUX1, ZmPIN1b and ZmPIN1c were down-regulated.The analysis of promoter sequences and expression patterns of these auxin polar transport genes indicated that these genes were involved in the morphogenesis of plant and the response to low phosphate stress.Introduced ZmPIN1a/1b or ZmAUX1 into maize enhanced the tolerance to low phosphate stress and modified the morphology of plantsBased on the analysis of auxin polar transporter, the sense and antisense constructs of ZmPIN1a, ZmPIN1b and ZmAUX1 were introduced into maize inbred line DH4866. The characteristics, yields, root architecture and their response to low phosphate of the transgenic homozygous lines were determined.Overexpression of ZmPIN1a or ZmPIN1b improved the maize root development, especially for the ZmPIN1a. The ZmPIN1a sense lines have distinctive root architecture with long seminal roots, high density lateral roots, but the length of lateral roots reduced, resulted in the increased root volume and changed root architecture. Biomass determination demonstrated that the root weight of ZmPIN1a sense lines were significantly higher than that of WT, while the shoot were lighter than WT. When cultured in SP solution the root system of ZmPIN1a sense lines were more developed, the number of lateral roots was 121～173% of WT, although the number of seminal roots were not significantly different with WT. At the same time the number of lateral roots of antisense lines was 82%-106% of WT plants. The analysis of root length showed that the length of seminal roots of ZmPIN1a sense lines were more longer than WT, but the average root length were lower than WT, resulted in a root system with long seminal root with more density lateral roots. When cultured in LP solution, all the lines behaved low phosphate acclimation, and showed the increased seminal root length and inhibited lateral root formation. Compare with WT and antisense lines, the ZmPIN1a sense lines were more distinct from others. The root number of sense plant was 141～261% of WT, and the total length of root was 98～132% of WT, this implied the overexpression of ZmPIN1a promoted the occurrence of lateral root, which might be a consequence of local auxin gradient change induced by the low phosphate stress. The sense lines showed the reduced height of plant and ear position, and the increased lateral root number and length of seminal root, and improved yield. These traits offered an excellent prospect in maize breeding.The dry weights of ZmPIN1b sense line were slightly higher than WT, but the dry weight of root was significantly higher than WT and antisense lines. The latter were similar to WT. When cultured in SP solution, the number of lateral roots was 129～146% of WT and the length of root system was 114%～138% of WT, while the antisense lines were slightly less than WT. When cultured in LP solution, the sense and antisense lines both behaved decreased low phosphate sensitivity and the number and length of lateral roots were not significantly different of plants in SP solution. It was possible that ZmPIN1b expression levels affected the acclimation to low phosphate stress of the plants.Overexpression of ZmAUX1 in maize could maintain relative higher biomass and better growth development under low phosphate conditions. Moreover, the overexpression of ZmA UX1 regulated the expression of other auxin polar transport related genes.These transgene plants were very impressed us that it was possible to improve the stress tolerance and produce high yield breeding by changing the root architecture via introducing the auxin polar transport genes into maize.The transcriptom changes by manipulating the expression level of ZmPINlaThe expression of auxin polar transporter genes in ZmPIN1a sense, antisense and WT plants were analysis by Real-time RT-PCR. We found that overexpression of ZmPIN1a enhanced the expression of ZmAUX1, ZmPIN1b, ZmPIN1c, ZmPIN3b and ZmPIN3b in the shoot and root of 4 day germinated seedlings, while in the antisense lines theses genes decreased with various degrees. The expression of ZmAUX1 in the sense lines were 3-6 fold of WT in the shoot and 1.4-2.2 fold in the root. The expression of ZmPIN1b was similar to ZmAUX1 in the root and 2-5 fold of WT in the shoot. The expression of ZmPIN1c was similar to ZmAUX1,but the fold was smaller than others. The expression of two PIN3 genes was induced by ZmPIN1a, but no significant parallelism in expression levels between ZmPIN1a and them.Used the DGEs, the transcriptions of root and shoot of ZmPIN1a sense, antisense lines and WT cultured in different Pi content solutions were analysis. The overexpression of ZmPIN1a affected largely the transpcripts of the plants compared with the antisense lines, especially in the root. Overexpression of ZmPIN1a led to significant changes of root architecture and plant development. Auxin and ethylene signaling were obviously modified in the ZmPINla sense line, and the remarkable change was the genes involved in photosynthesis were up-regulated on transcribe level. Moreover, many metabolic intermediate might be accumulated in the sense line and the circadian rhythm related factors were differentially expressed in different lines. These provided abundant information to explore the relationships among auxin-root architecture-low phosphate.In summary, we have examined the gene expression profiles of maize plants from inbred Q319 and its analogical line 99038 using an Oligonucleotide Array; Some important signaling pathway and key genes related to low phosphate response and root morphology were characterized. Thirteen auxin polar transporter genes were cloned and examined for their expression profiles and promoter sequence. Using Agrobacterium mediated maize shoot transformation, the sense and antisense ZmPTF1, Zea mays auxin transporter protein 3 (ZmAUX1), ZmPIN1a and ZmPIN1b were intruded into maize inbred line. The morphology, development, yield, root architecture and their response to low phosphate stress of the transgene lines were determined. We have obtained the overexpression lines of ZmPTFl or ZmPIN1a which have well prospect in maize breeding. |
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