The poplar basic helix-loop-helix transcription factor BEE3 - Like gene affects biomass production by enhancing proliferation of xylem cells in poplar.

Brassinosteroids (BRs) play important roles in many aspects of plant growth and development, including regulation of vascular cambium activities and cell elongation. BR-induced BEE3 (brassinosteroid enhanced expression 3) is required for a proper BR response. Here, we identified a poplar (Populus alba × Populus glandulosa) BEE3-like gene, PagBEE3L, encoding a putative basic helix-loop-helix (bHLH)-type transcription factor. Expression of PagBEE3L was induced by brassinolide (BL). Transcripts of PagBEE3L were mainly detected in stems, with the internode having a low level of transcription and the node having a relatively higher level. The function of the PagBEE3L gene was investigated through phenotypic analyses with PagBEE3L-overexpressing (ox) transgenic lines. This work particularly focused on a potential role of PagBEE3L in stem growth and development of polar. The PagBEE3L-ox poplar showed thicker and longer stems than wild-type plants. The xylem cells from the stems of PagBEE3L-ox plants revealed remarkably enhanced proliferation, resulting in an earlier thickening growth than wild-type plants. Therefore, this work suggests that xylem development of poplar is accelerated in PagBEE3L-ox plants and PagBEE3L plays a role in stem growth by increasing the proliferation of xylem cells to promote the initial thickening growth of poplar stems.

Overexpression of sweetpotato expansin cDNA (IbEXP1) increases seed yield in Arabidopsis.

Results of transcriptome analyses suggest that expansin genes play an active role in seed development and yield, but gain- or loss-of-function studies have not yet elucidated the functional role(s) of the expansin gene(s) in these processes. We have overexpressed a sweetpotato expansin gene (IbEXP1) in Arabidopsis under the control of cauliflower mosaic 35S promoter in an attempt to determine the effect of the expansin gene in seed development and yield in heterologous plants. The growth rate was enhanced in IbEXP1-overexpressing (ox) plants relative to wild-type Col-0 plants during early vegetative growth stage. At the reproductive stage, the number of rosette leaves was higher in IbEXP1-ox plants than that in Col-0 plants, and siliques were thicker. IbEXP1-ox plants produced larger seeds, accumulated more protein and starch in each seed, and produced more inflorescence stems and siliques than Col-0 plants, leading to a 2.1-2.5-fold increase in total seed yield per plant. The transcript level of IbEXP1 was up-regulated in response to brassinosteroid (BR) treatment in sweetpotato, and the transcript levels of three BR-responsive genes, fatty acid elongase 3-ketoacyl-CoA synthase 1, HAIKU1 and MINISEED3, were also increased in IbEXP1-ox Arabidopsis plants, suggesting a possible involvement of IbEXP1 in at least one of the BR signaling pathways. Based on these results, we suggest that overexpression of IbEXP1 gene in heterologous plants is effective in increasing seed size and number and, consequently, seed yield.

Down-regulation of the IbEXP1 gene enhanced storage root development in sweetpotato.

The role of an expansin gene (IbEXP1) in the formation of the storage root (SR) was investigated by expression pattern analysis and characterization of IbEXP1-antisense sweetpotato (Ipomoea batatas cv. Yulmi) plants in an attempt to elucidate the molecular mechanism underlying SR development in sweetpotato. The transcript level of IbEXP1 was high in the fibrous root (FR) and petiole at the FR stage, but decreased significantly at the young storage root (YSR) stage. IbEXP1-antisense plants cultured in vitro produced FRs which were both thicker and shorter than those of wild-type (WT) plants. Elongation growth of the epidermal cells was significantly reduced, and metaxylem and cambium cell proliferation was markedly enhanced in the FRs of IbEXP1-antisense plants, resulting in an earlier thickening growth in these plants relative to WT plants. There was a marked reduction in the lignification of the central stele of the FRs of the IbEXP1-antisense plants, suggesting that the FRs of the mutant plants possessed a higher potential than those of WT plants to develop into SRs. IbEXP1-antisense plants cultured in soil produced a larger number of SRs and, consequently, total SR weight per IbEXP1-antisense plant was greater than that per WT plant. These results demonstrate that SR development was accelerated in IbEXP1-antisense plants and suggest that IbEXP1 plays a negative role in the formation of SR by suppressing the proliferation of metaxylem and cambium cells to inhibit the initial thickening growth of SRs. IbEXP1 is the first sweetpotato gene whose role in SR development has been directly identified in soil-grown transgenic sweetpotato plants.

A sweetpotato SRD1 promoter confers strong root-, taproot-, and tuber-specific expression in Arabidopsis, carrot, and potato.

Harvestable, starch-storing organs of plants, such as fleshy taproots and tubers, are important agronomic products that are also suitable target organs for use in the molecular farming of recombinant proteins due to their strong sink strength. To exploit a promoter directing strong expression restricted to these storage organs, we isolated the promoter region (3.0 kb) of SRD1 from sweetpotato (Ipomoea batatas cv. 'White Star') and characterized its activity in transgenic Arabidopsis, carrot, and potato using the ß-glucuronidase (GUS) gene (uidA) as a reporter gene. The SRD1 promoter conferred root-specific expression in transgenic Arabidopsis, with SRD1 promoter activity increasing in response to exogenous IAA. A time-course study of the effect of IAA (50 µM) revealed a maximum increase in SRD1 promoter activity at 24 h post-treatment initiation. A serial 5' deletion analysis of the SRD1 promoter identified regions related to IAA-inducible expression as well as regions containing positive and negative elements, respectively, controlling the expression level. In transgenic carrot, the SRD1 promoter mediated strong taproot-specific expression, as evidenced by GUS staining being strong in almost the entire taproot, including secondary phloem, secondary xylem and vascular cambium. The activity of the SRD1 promoter gradually increased with increasing diameter of the taproot in the transgenic carrot and was 10.71-fold higher than that of the CaMV35S promoter. The SRD1 promoter also directed strong tuber-specific expression in transgenic potato. Taken together, these results demonstrate that the SRD1 promoter directs strong expression restricted to the underground storage organs, such as fleshy taproots and tubers, as well as fibrous root tissues.

SRD1 is involved in the auxin-mediated initial thickening growth of storage root by enhancing proliferation of metaxylem and cambium cells in sweetpotato (Ipomoea batatas).

A sweetpotato (Ipomoea batatas cv. 'Jinhongmi') MADS-box protein cDNA (SRD1) has been isolated from an early stage storage root cDNA library. The role of the SRD1 gene in the formation of the storage root in sweetpotato was investigated by an expression pattern analysis and characterization of SRD1-overexpressing (ox) transgenic sweetpotato plants. Transcripts of SRD1 were detected only in root tissues, with the fibrous root having low levels of the transcript and the young storage root showing relatively higher transcript levels. SRD1 mRNA was mainly found in the actively dividing cells, including the vascular and cambium cells of the young storage root. The transcript level of SRD1 in the fibrous roots increased in response to 1000 muM indole-3-acetic acid (IAA) applied exogenously. During the early stage of storage root development, the endogenous IAA content and SRD1 transcript level increased concomitantly, suggesting an involvement of SRD1 during the early stage of the auxin-dependent development of the storage root. SRD1-ox sweetpotato plants cultured in vitro produced thicker and shorter fibrous roots than wild-type plants. The metaxylem and cambium cells of the fibrous roots of SRD1-ox plants showed markedly enhanced proliferation, resulting in the fibrous roots of these plants showing an earlier thickening growth than those of wild-type plants. Taken together, these results demonstrate that SRD1 plays a role in the formation of storage roots by activating the proliferation of cambium and metaxylem cells to induce the initial thickening growth of storage roots in an auxin-dependent manner.

Two sweetpotato ADP-glucose pyrophosphorylase isoforms are regulated antagonistically in response to sucrose content in storage roots.

The transcriptional regulation of ADP-glucose pyrophosphorylase (AGPase) genes in detached leaves in response to exogenous sucrose has been investigated earlier; however the effects of endogenous sucrose on AGPase gene transcription in leaves or starch-accumulating tissues have not yet been determined. We therefore have investigated the relationship between endogenous sucrose content in the storage tissues of sweetpotato (Ipomoea batatas cv. Yulmi) and the rate of transcription of the two sweetpotato AGPase isoforms, ibAGP1 and ibAGP2, by means of transient expression analysis of their promoters. Sequence analysis of the two promoters identified putative sucrose-responsive elements on the ibAGP1 promoter and, conversely, putative sucrose-starvation elements on the ibAGP2 promoter. Transient expression analyses on transverse storage root sections revealed that the ibAGP1 and ibAGP2 promoters directed strong expression in the sweetpotato storage roots (diameter: 1.5 cm). Sucrose contents of the sweetpotato storage roots were positively correlated with growth of the storage root. In the storage roots, ibAGP1 promoter activity became stronger with increasing endogenous sucrose levels, while ibAGP2 promoter activity became markedly weaker. Consequently, ibAGP2 was expressed primarily during the early stages of storage root development, whereas ibAGP1 was abundantly expressed in the later stages, during which a profound level of starch accumulation occurs. The antagonistic regulation of the two promoters in response to endogenous sucrose levels was also confirmed in carrot (Daucus carota L. cv. Hapa-ochon) taproots.

Genomic organizations of two small subunit ADP-glucose pyrophosphorylase genes from sweetpotato.

The genomic features of the small subunit ADP-glucose pyrophosphorylase (AGPase) isoforms are different in barley and maize. The two isoforms found in barley originated from one single gene through alternative splicing, while two independent genes encode the two isoforms in maize. To ascertain the genomic organizations of two small subunit AGPase isoforms in sweetpotato (ibAGP1 and ibAGP2), we isolated genomic DNAs containing the entire coding regions of two genes. Complete genomic structures of ibAGP1 and ibAGP2 were ascertained by the sequencing of these genomic regions. The transcribed regions of ibAGP1 and ibAGP2, comprising nine exons and eight introns, were distributed over 3.9 and 4.0 kb, respectively. The eight introns differed in length, from 76 to 946 bp in ibAGP1, and from 76 to 811 bp in ibAGP2, while the locations of introns in ibAGP1 and ibAGP2 were identical. There was 46-58% sequence identity between the intron sequences of the two genes. Intron sequence analyses suggested that either duplication in each intron, or gene conversion between introns of two isoforms, might cause major intron size differences between the two genes. Altogether, these results indicate that two small subunit AGPase isoforms in sweetpotato are encoded by two independent genes, in a fashion similar to that of maize small subunit AGPase genes.