Aiqun Chen

Identification of two conserved cis‐acting elements, MYCS and P1BS, involved in the regulation of mycorrhiza‐activated phosphate transporters in eudicot species

• In this study, six putative promoter regions of phosphate transporter Pht1;3, Pht1;4 and Pht1;5 genes were isolated from eggplant and tobacco using the inverse polymerase chain reaction (iPCR). The isolated sequences show evolutionary conservation and divergence within/between the two groups of Pht1;3 and Pht1;4/Pht1;5. • Histochemical analyses showed that all six promoter fragments were sufficient to drive β-glucuronidase (GUS) expression specifically in arbuscular mycorrhizal (AM) tobacco roots and were confined to distinct cells containing AM fungal structures (arbuscules or intracellular hyphae). • A series of promoter truncation and mutation analyses combined with phylogenetic footprinting of these promoters revealed that at least two cis-regulatory elements--the mycorrhiza transcription factor binding sequence (MYCS) first identified in this study and P1BS--mediated the transcriptional activation of the AM-mediated inorganic phosphate (Pi) transporter genes. Deletion or partial mutation of either of the two motifs in the promoters could cause a remarkable decrease, or even complete absence, of the promoter activity. • Our results propose that uptake of inorganic phosphate (Pi) by AM fungi is regulated, at least partially, in an MYCS- and P1BS-dependent manner in eudicot species. Our finding offers new insights into the molecular mechanisms underlying the coordination between the AM and the Pi signalling pathways.

Analyses of the sucrose synthase gene family in cotton: structure, phylogeny and expression patterns

BackgroundIn plants, sucrose synthase (Sus) is widely considered as a key enzyme involved in sucrose metabolism. Several paralogous genes encoding different isozymes of Sus have been identified and characterized in multiple plant genomes, while limited information of Sus genes is available to date for cotton.ResultsHere, we report the molecular cloning, structural organization, phylogenetic evolution and expression profiles of seven Sus genes (GaSus1 to 7) identified from diploid fiber cotton (Gossypium arboreum). Comparisons between cDNA and genomic sequences revealed that the cotton GaSus genes were interrupted by multiple introns. Comparative screening of introns in homologous genes demonstrated that the number and position of Sus introns are highly conserved among Sus genes in cotton and other more distantly related plant species. Phylogenetic analysis showed that GaSus1, GaSus2, GaSus3, GaSus4 and GaSus5 could be clustered together into a dicot Sus group, while GaSus6 and GaSus7 were separated evenly into other two groups, with members from both dicot and monocot species. Expression profiles analyses of the seven Sus genes indicated that except GaSus2, of which the transcripts was undetectable in all tissues examined, and GaSus7, which was only expressed in stem and petal, the other five paralogues were differentially expressed in a wide ranges of tissues, and showed development-dependent expression profiles in cotton fiber cells.ConclusionsThis is a comprehensive study of the Sus gene family in cotton plant. The results presented in this work provide new insights into the evolutionary conservation and sub-functional divergence of the cotton Sus gene family in response to cotton fiber growth and development.

Involvement of OsPht1;4 in phosphate acquisition and mobilization facilitates embryo development in rice

Phosphate (Pi) transporters mediate acquisition and transportation of Pi within plants. Here, we investigated the functions of OsPht1;4 (OsPT4), one of the 13 members of the Pht1 family in rice. Quantitative real-time RT-PCR analysis revealed strong expression of OsPT4 in roots and embryos, and OsPT4 promoter analysis using reporter genes confirmed these findings. Analysis using rice protoplasts showed that OsPT4 localized to the plasma membrane. OsPT4 complemented a yeast mutant defective in Pi uptake, and also facilitated increased accumulation of Pi in Xenopus oocytes. Further, OsPT4 genetically modified (GM) rice lines were generated by knockout/knockdown or over-expression of OsPT4. Pi concentrations in roots and shoots were significantly lower and higher in knockout/knockdown and over-expressing plants, respectively, compared to wild-type under various Pi regimes. (33) Pi uptake translocation assays corroborated the altered acquisition and mobilization of Pi in OsPT4 GM plants. We also observed effects of altered expression levels of OsPT4 in GM plants on the concentration of Pi, the size of the embryo, and several attributes related to seed development. Overall, our results suggest that OsPT4 encodes a plasma membrane-localized Pi transporter that facilitates acquisition and mobilization of Pi, and also plays an important role in development of the embryo in rice.

Genome-wide investigation and expression analysis suggest diverse roles and genetic redundancy of Pht1 family genes in response to Pi deficiency in tomato

BackgroundPhosphorus (P) deficiency is one of the major nutrient stresses limiting plant growth. The uptake of P by plants is well considered to be mediated by a number of high-affinity phosphate (Pi) transporters belonging to the Pht1 family. Although the Pht1 genes have been extensively identified in several plant species, there is a lack of systematic analysis of the Pht1 gene family in any solanaceous species thus far.ResultsHere, we report the genome-wide analysis, phylogenetic evolution and expression patterns of the Pht1 genes in tomato (Solanum lycopersicum). A total of eight putative Pht1 genes (LePT1 to 8), distributed on three chromosomes (3, 6 and 9), were identified through extensive searches of the released tomato genome sequence database. Chromosomal organization and phylogenetic tree analysis suggested that the six Pht1 paralogues, LePT1/3, LePT2/6 and LePT4/5, which were assigned into three pairs with very close physical distance, were produced from recent tandem duplication events that occurred after Solanaceae splitting with other dicot families. Expression analysis of these Pht1 members revealed that except LePT8, of which the transcript was undetectable in all tissues, the other seven paralogues showed differential but partial-overlapping expression patterns. LePT1 and LePT7 were ubiquitously expressed in all tissues examined, and their transcripts were induced abundantly in response to Pi starvation; LePT2 and LePT6, the two paralogues harboring identical coding sequence, were predominantly expressed in Pi-deficient roots; LePT3, LePT4 and LePT5 were strongly activated in the roots colonized by arbuscular mycorrhizal fungi under low-P, but not high-P condition. Histochemical analysis revealed that a 1250-bp LePT3 promoter fragment and a 471-bp LePT5 promoter fragment containing the two elements, MYCS and P1BS, were sufficient to direct the GUS reporter expression in mycorrhizal roots and were limited to distinct cells harboring AM fungal structures. Additionally, the four paralogues, LePT1, LePT2, LePT6 and LePT7, were very significantly down-regulated in the mycorrhizal roots under low Pi supply condition.ConclusionsThe results obtained from this study provide new insights into the evolutionary expansion, functional divergence and genetic redundancy of the Pht1 genes in response to Pi deficiency and mycorrhizal symbiosis in tomato.

Improving rice tolerance to potassium deficiency by enhancing OsHAK16p:WOX11-controlled root development

Potassium (K) deficiency in plants confines root growth and decreases root-to-shoot ratio, thus limiting root K acquisition in culture medium. A WUSCHEL-related homeobox (WOX) gene, WOX11, has been reported as an integrator of auxin and cytokinin signalling that regulates root cell proliferation. Here, we report that ectopic expression of WOX11 gene driven by the promoter of OsHAK16 encoding a low-K-enhanced K transporter led to an extensive root system and adventitious roots and more effective tiller numbers in rice. The WOX11-regulated root and shoot phenotypes in the OsHAK16p:WOX11 transgenic lines were supported by K-deficiency-enhanced expression of several RR genes encoding type-A cytokinin-responsive regulators, PIN genes encoding auxin transporters and Aux/IAA genes. In comparison with WT, the transgenic lines showed increases in root biomass, root activity and K concentrations in the whole plants, and higher soluble sugar concentrations in roots particularly under low K supply condition. The improvement of sugar partitioning to the roots by the expression of OsHAK16p:WOX11 was further indicated by increasing the expression of OsSUT1 and OsSUT4 genes in leaf blades and several OsMSTs genes in roots. Expression of OsHAK16p:WOX11 in the rice grown in moderate K-deficient soil increased total K uptake by 72% and grain yield by 24%-32%. The results suggest that enlarging root growth and development by the expression of WOX11 in roots could provide a useful option for increasing K acquisition efficiency and cereal crop productivity in low K soil.

Identification of microRNAs in six solanaceous plants and their potential link with phosphate and mycorrhizal signaling

To date, only a limited number of solanaceous miRNAs have been deposited in the miRNA database. Here, genome-wide bioinformatic identification of miRNAs was performed in six solanaceous plants (potato, tomato, tobacco, eggplant, pepper, and petunia). A total of 2,239 miRNAs were identified following a range of criteria, of which 982 were from potato, 496 from tomato, 655 from tobacco, 46 from eggplant, 45 were from pepper, and 15 from petunia. The sizes of miRNA families and miRNA precursor length differ in all the species. Accordingly, 620 targets were predicted, which could be functionally classified as transcription factors, metabolic enzymes, RNA and protein processing proteins, and other proteins for plant growth and development. We also showed evidence for miRNA clusters and sense and antisense miRNAs. Additionally, five Pi starvation- and one arbuscular mycorrhiza (AM)-related cis-elements were found widely distributed in the putative promoter regions of the miRNA genes. Selected miRNAs were classified into three groups based on the presence or absence of P1BS and MYCS cis-elements, and their expression in response to Pi starvation and AM symbiosis was validated by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). These results show that conserved miRNAs exist in solanaceous species and they might play pivotal roles in plant growth, development, and stress responses.

How does phosphate status influence the development of the arbuscular mycorrhizal symbiosis

Most terrestrial plant roots form mutualistic symbiosis with soil-borne arbuscular mycorrhizal fungi (AMF), a characteristic feature of which is nutrient exchange between the two symbiotic partners. Phosphate (Pi) is the main benefit the host plants acquired from the AMF. It has long been a common realization that high Pi supply could suppress the AMF development. However, the direct molecular regulatory mechanisms underlying this plant directed suppression are lacking. Here, we reviewed the recent work providing the evidences that high Pi supply induces transcriptional alteration, leading to the inhibition of AMF development at different stages of AM symbiosis, and gave our view on potential cross-talk among Pi starvation, AM as well as phytohormone signaling.

The Characterization of Six Auxin-Induced Tomato GH3 Genes Uncovers a Member, SlGH3.4, Strongly Responsive to Arbuscular Mycorrhizal Symbiosis

In plants, the GH3 gene family is widely considered to be involved in a broad range of plant physiological processes, through modulation of hormonal homeostasis. Multiple GH3 genes have been functionally characterized in several plant species; however, to date, limited works to study the GH3 genes in tomato have been reported. Here, we characterize the expression and regulatory profiles of six tomato GH3 genes, SlGH3.2, SlGH3.3, SlGH3.4, SlGH3.7, SlGH3.9 and SlGH3.15, in response to different phytohormone applications and arbuscular mycorrhizal (AM) fungal colonization. All six GH3 genes showed inducible responses to external IAA, and three members were significantly up-regulated in response to AM symbiosis. In particular, SlGH3.4, the transcripts of which were barely detectable under normal growth conditions, was strongly activated in the IAA-treated and AM fungal-colonized roots. A comparison of the SlGH3.4 expression in wild-type plants and M161, a mutant with a defect in AM symbiosis, confirmed that SlGH3.4 expression is highly correlated to mycorrhizal colonization. Histochemical staining demonstrated that a 2,258 bp SlGH3.4 promoter fragment could drive β-glucuronidase (GUS) expression strongly in root tips, steles and cortical cells of IAA-treated roots, but predominantly in the fungal-colonized cells of mycorrhizal roots. A truncated 654 bp promoter failed to direct GUS expression in IAA-treated roots, but maintained the symbiosis-induced activity in mycorrhizal roots. In summary, our results suggest that a mycorrhizal signaling pathway that is at least partially independent of the auxin signaling pathway has evolved for the co-regulation of the auxin- and mycorrhiza-activated GH3 genes in plants.

Tomato sugar transporter genes associated with mycorrhiza and phosphate

In arbuscular mycorrhizal (AM) symbiosis, there is a reciprocal nutrient exchange between symbiotic partners. AM fungi assist the plants with uptake of mineral nutrients from the soil, especially phosphate (Pi). The host plants, in return, provide the fungi with sugar. In contrast to the studies on the symbiotic transport of Pi in arbuscular mycorrhiza, there have been few investigations devoted specifically to sugar transport in AM symbiosis. Using reverse transcriptase PCR (RT-PCR), we analyzed the effects of mycorrhiza and Pi on the expression of seven putative sugar transporter genes in tomato plants. The expression of LeSUT1 and LeHT2, classified, respectively, as putative sucrose and hexose transporter genes, were down-regulated in the roots inoculated with AM fungi Glomus caledonium or Glomus intraradices. Unexpectedly, the expression of LeST3, a putative monosaccharide transporter gene, was decreased by inoculation of Glomus caledonium, but was enhanced by inoculation of Glomus intraradices in both the roots and leaves. High-Pi in the tomato decreased the expression of LeHT2 and LeST3 in the roots, and enhanced that of LeHT2 in the leaves. The contradictory regulation of the expression of LeST3 in the AM symbiosis indicates that different interaction may take place during mycorrhizal colonization, and a quite complicated regulation of expression of the sugar transporter genes exist in mycorrhizal tomato.

Analysis of tomato plasma membrane H + -ATPase gene family suggests a mycorrhiza-mediated regulatory mechanism conserved in diverse plant species

In plants, the plasma membrane H+-ATPase (HA) is considered to play a crucial role in regulating plant growth and respoding to environment stresses. Multiple paralogous genes encoding different isozymes of HA have been identified and characterized in several model plants, while limited information of the HA gene family is available to date for tomato. Here, we describe the molecular and expression features of eight HA-encoding genes (SlHA1-8) from tomato. All these genes are interrupted by multiple introns with conserved positions. SlHA1, 2, and 4 were widely expressed in all tissues, while SlHA5, 6, and 7 were almost only expressed in flowers. SlHA8, the transcripts of which were barely detectable under normal or nutrient-/salt-stress growth conditions, was strongly activated in arbuscular mycorrhizal (AM) fungal-colonized roots. Extreme lack of SlHA8 expression in M161, a mutant defective to AM fungal colonization, provided genetic evidence towards the dependence of its expression on AM symbiosis. A 1521-bp SlHA8 promoter could direct the GUS reporter expression specifically in colonized cells of transgenic tobacco, soybean, and rice mycorrhizal roots. Promoter deletion assay revealed a 223-bp promoter fragment of SlHA8 containing a variant of AM-specific cis-element MYCS (vMYCS) sufficient to confer the AM-induced activity. Targeted deletion of this motif in the corresponding promoter region causes complete abolishment of GUS staining in mycorrhizal roots. Together, these results lend cogent evidence towards the evolutionary conservation of a potential regulatory mechanism mediating the activation of AM-responsive HA genes in diverse mycorrhizal plant species.