NIP1;2 is a plasma membrane-localized transporter mediating aluminum uptake, translocation, and tolerance in Arabidopsis.

Members of the aquaporin (AQP) family have been suggested to transport aluminum (Al) in plants; however, the Al form transported by AQPs and the roles of AQPs in Al tolerance remain elusive. Here we report that NIP1;2, a plasma membrane-localized member of the Arabidopsis nodulin 26-like intrinsic protein (NIP) subfamily of the AQP family, facilitates Al-malate transport from the root cell wall into the root symplasm, with subsequent Al xylem loading and root-to-shoot translocation, which are critical steps in an internal Al tolerance mechanism in Arabidopsis We found that NIP1;2 transcripts are expressed mainly in the root tips, and that this expression is enhanced by Al but not by other metal stresses. Mutations in NIP1;2 lead to hyperaccumulation of toxic Al3+ in the root cell wall, inhibition of root-to-shoot Al translocation, and a significant reduction in Al tolerance. NIP1;2 facilitates the transport of Al-malate, but not Al3+ ions, in both yeast and Arabidopsis We demonstrate that the formation of the Al-malate complex in the root tip apoplast is a prerequisite for NIP1;2-mediated Al removal from the root cell wall, and that this requires a functional root malate exudation system mediated by the Al-activated malate transporter, ALMT1. Taken together, these findings reveal a critical linkage between the previously identified Al exclusion mechanism based on root malate release and an internal Al tolerance mechanism identified here through the coordinated function of NIP1;2 and ALMT1, which is required for Al removal from the root cell wall, root-to-shoot Al translocation, and overall Al tolerance in Arabidopsis.

Sequential amplification of flanking sequences by Y-shaped adaptor dependent extension using multiple templates.

Y-shaped adaptor dependent extension (YADE) method is a useful tool to amplify the flanking sequence of a known DNA sequence, but its efficiency is frequently limited by the restriction sites around the known sequence. In this paper, we demonstrated that using multiple templates derived from several restrictions and ligations could dramatically increase the efficiency of YADE method and render it suitable for sequential amplification of flanking sequences. With templates originating from 7 digestions, a 2,228-bp 5'-upstream sequence of a cotton small GTPase gene was obtained by two rounds of sequential YADE amplifications. The results demonstrated that the YADE method with multiple templates may be a useful tool for sequential PCR walking in complex genomes.

Functional expression of the cotton gibberellic acid oxidase homologous gene GhGA20ox1 in tobacco.

To determine the physiological function of GhGA20ox1, a homologous gene of GA 20-oxidase from elongating cotton fibers, we expressed this gene ectopically in Nicotiana benthamiana. Reverse transcription-PCR analysis showed that the GhGA20ox1 gene was expressed in the transgenic plants at various levels. It was demonstrated that overexpression of GhGA20ox1 enhanced preferentially the GA(4+7) biosynthesis in N. benthamiana and conferred GA-overproduction characters to transformants. The extent of phenotypic alteration in the transgenic plants was found to correlate with the transcriptional levels of GhGA20ox1 and GA contents. Results indicated that the GhGA20ox1 gene promoted the biosynthesis of the active GAs (GA(4+7)) in transgenic tobacco plants therefore represents a useful gene for manipulating GA levels.

[Cloning and expression analysis of two Rac genes from cotton (Gossypium hirsutum L.)].

Plant Rac proteins belong to an important group of signal switches anchoring on membranes, involved in various physiological processes including cell polar growth, synthesis of secondary wall, resistance response and hormone signaling. In the attempt to elucidate the molecular mechanism of initiation and elongation of cotton fiber, two cotton Rac protein genes, designated as GhRacA and GhRacB, were amplified from elongating fibers and cloned. It was demonstrated that, the cDNA of GhRacA contained 959 bp and encoded a putative polypepetide of 211 aa, while GhRacB was 920 bp in length, encoding a predicted protein of 195 aa. These two cotton Rac proteins, GhRacA and GhRacB, contained conserved regions involved in GTP/GDP binding and activation, an effector region and a polybasic region. A conserved prenylation site CSIL was found in GhRacB, while no apparent prenylation site was discovered in GhRacA. Sequence comparisons showed that GhRacA and GhRacB were two novel Rac proteins from cotton. The expression patterns of GhRacA and GhRacB was analyzed by RT-PCR. It was demonstrated that these two Rac protein genes were both expressed in root, hypocotyls, stem, leaf and fibers, and the highest level of transcripts was to accumulate in the fibers at the stage of initiation and elongation, suggesting that the two Rac genes, GhRacA and GhRacB, might play an important role in the early stage of fiber development.

Moderately enhancing cytokinin level by down-regulation of GhCKX expression in cotton concurrently increases fiber and seed yield.

Cotton is the leading natural fiber crop in the world. Cotton seeds are also an important oil and protein source. However, enhancement of fiber abundance usually leads to a smaller seed. Thus, it has become a challenge for cotton breeding to concurrently increase fiber yield and seed yield. To improve cotton yield, we elevated the endogenous cytokinin level in transgenic cotton by constitutive suppression of cytokinin dehydrogenase (CKX), a key negative regulator controlling endogenous cytokinin in plants. The slightly and moderately suppressed transgenic cotton plants showed normal growth and development, while the severely suppressed plants exhibited a typical cytokinin-overproduction alteration. The suppression of CKX led to an enhancement of endogenous cytokinins in transgenic cotton plants. Total cytokinins in moderately suppressed lines, CR-3 and CR-6, increased by 20.4 and 55.5 % respectively, and that in the severely suppressed line (CR-13) increased by 134.2 % compared to the wild type. The moderately suppressed lines showed a delay in leaf senescence, higher photosynthesis, more fruiting branches and bolls, and bigger seed size. Field trials showed that seed yield and lint yield of the moderately suppressed CR-6 line increased by 15.4 and 20.0 %, respectively. Meanwhile, the enhanced cytokinin level in transgenic cottons did not show significant influence on fiber qualities. Our data demonstrated that CKX is a promising gene for crop yield improvement.

[Cloning and expression analysis of a LIM-domain protein gene from cotton (Gossypium hirsuturm L.)].

LIM-domain protein plays an important role in various cellular processes, including construction of cytoskeleton, transcription control and signal transduction. Based on cotton fiber EST database and contig analysis, the coding region of a cotton LIM-domain protein gene (GhLIM1) was obtained by RT-PCR from 4DPA (day post anthesis) ovule with fiber. The cloned fragment of 848 bp contains an open reading frame of 570 bp, coding for a polypeptide of 189 amino acids. It was demonstrated that the deduced GhLIM1 protein was highly homologous to the LIM-domain protein of sunflower (Helianthus annuus), tobacco (Nicotiana tabacum) and Arabidopsis thaliana. Two intact LIM-domains, with the conserved sequence of a double zinc-finger structure (C-X2-C-X17-19-H-X2-C-X2-C-X2-C-X16-24-C-X2-H), were found in the GhLIM1 protein. RT-PCR and Northern blot analysis showed that GhLIM1 gene expressed in root, shoot tip, hypocotyls, bud, leaf, anther, ovule and fiber (4DPA, 12DPA, 18DPA). However it was preferentially expressed in the shoot tip, fiber and ovule. It was proposed that the express of GhLIM1 gene is related to cotton fiber development.

[Cloning and characterization of the PTS2 receptor gene (GhPex7) from cotton (Gossypium hirsutum L.)].

Peroxisomal targeting signals (PTS), including PTS1 and PTS2, were proposed to play an important role in introducing proteins into the peroxisomal matrix. Pex7, the PTS2 receptor, is crucial to the import of PTS2 proteins. Based on the sequence of a fragment (F010) recovered from cDNA-AFLP, the full-length cDNA sequence was obtained by RACE and the contig in cotton ESTs, and the coding sequence was further cloned. The GhPex7 cDNA, 1314 bp in length, contained 5 non-coding (77 bp) upstream, 3 complete downstream with polyA signal tail and ORF of 954 bp, which coded for a deduced protein of 317 amino acids. The deduced protein had a predicted MW 35.57 kDa and a pI of 5.603. Homology analysis demonstrated that GhPex7 protein contained three highly conserved domains and three G-bata domains of WD-40 proteins family. The sequence of nucleotides and amino acids shared 83% and 76% identity with known Arabidopsis AtPex7, respectively, and its amino acid sequence shared from 28% to 42% identity with those of Drosophila melanogaster, Saccach, cerevisiae, Mus musculus and Homo sapiens. Southern blotting suggested that at least two copies of GhPex7 gene existed in Gossypium hirstum genome. By Northern blotting and RT-PCR analysis, expression of the GhPex7 was detected in roots, stems, leaves, buds, ovules and fibers, however it was stronger in leaves and stems than in other tissues. At the stage of cotton's ovules and fibers development, RT-PCR analysis also indicated that expression activity of GhPex7 in ovule at 0DPA mutant plant (no-fiber) was stronger than in wild type plant, its expression in wild type fiber at 23DPA was stronger than at 12, 16 DPA too.

[Prediction of yield and yield components in hybrid rice by using molecular markers].

Yield and yield components in hybrid rice were investigated using AFLP, RAPD and SSR markers. Ten restorer and five male-sterile lines were crossed in all possible pairs resulting in 50 crosses. Positive loci, effect-increasing loci, effect-decreasing loci and non-environmental loci were selected from the 931 marker loci surveyed in the 15 parental lines and their correlation with yield and yield components were analyzed. The results indicated as follows (1) The correlation between genetic difference and yield and yield components calculated on all three molecular loci failed to reach significant level for most of the traits investigated and can not be used to predict yield and yield components directly. (2) Positive loci were of limited usefulness in the prediction of yield and yield components for their variation with different traits investigated despite that they can improve the correlation coefficient in some degree. (3) Effect-increasing and effect-decreasing loci can greatly improve correlation coefficient and may be used to predict the yield and yield components for their consistence with the environment. (4) The coefficient based on non-environmental loci was high though it was a bit lower than that based on effect-increasing and effect-decreasing loci. It indicated that environment had great effect on yield and yield components in rice.

Gibberellin overproduction promotes sucrose synthase expression and secondary cell wall deposition in cotton fibers.

Bioactive gibberellins (GAs) comprise an important class of natural plant growth regulators and play essential roles in cotton fiber development. To date, the molecular base of GAs' functions in fiber development is largely unclear. To address this question, the endogenous bioactive GA levels in cotton developing fibers were elevated by specifically up-regulating GA 20-oxidase and suppressing GA 2-oxidase via transgenic methods. Higher GA levels in transgenic cotton fibers significantly increased micronaire values, 1000-fiber weight, cell wall thickness and cellulose contents of mature fibers. Quantitative RT-PCR and biochemical analysis revealed that the transcription of sucrose synthase gene GhSusA1 and sucrose synthase activities were significantly enhanced in GA overproducing transgenic fibers, compared to the wild-type cotton. In addition, exogenous application of bioactive GA could promote GhSusA1 expression in cultured fibers, as well as in cotton hypocotyls. Our results suggested that bioactive GAs promoted secondary cell wall deposition in cotton fibers by enhancing sucrose synthase expression.

Spatiotemporal manipulation of auxin biosynthesis in cotton ovule epidermal cells enhances fiber yield and quality.

The capacity of conventional breeding to simultaneously improve the yield and quality of cotton fiber is limited. The accumulation of the plant hormone indole-3-acetic acid (IAA) in cotton fiber initials prompted us to investigate the effects of genetically engineering increased IAA levels in the ovule epidermis. Targeted expression of the IAA biosynthetic gene iaaM, driven by the promoter of the petunia MADS box gene Floral Binding protein 7 (FBP7), increased IAA levels in the epidermis of cotton ovules at the fiber initiation stage. This substantially increased the number of lint fibers, an effect that was confirmed in a 4-year field trial. The lint percentage of the transgenic cotton, an important component of fiber yield, was consistently higher in our transgenic plants than in nontransgenic controls, resulting in a >15% increase in lint yield. Fiber fineness was also notably improved.

Gibberellin 20-oxidase promotes initiation and elongation of cotton fibers by regulating gibberellin synthesis.

Cotton is the leading natural fiber, and gibberellin (GA) is a phytohormone involved in the development of cotton fibers. However, it is largely unknown how the GA content in ovules and fibers is regulated and how the endogenous GA concentration affects fiber development. To address these questions, three GA 20-oxidase homologous genes (GhGA20ox1-3) were cloned and the endogenous bioactive GA content in developing ovules and fibers determined by liquid chromatography-electrospray ionization-mass spectrometry. Real-time reverse transcription PCR (RT-PCR) revealed that GhGA20ox1 expressed preferentially in elongating fibers and that the expression level varied with the endogenous GA content consistently, while GhGA20ox2 and GhGA20ox3 transcripts accumulated mainly in ovules. The GA accumulation kinetics as well as the GhGA20ox expression differed in ovules and the attached fibers, suggesting relatively independent GA regulation system in these two sites. Transgenic cotton, over-expressing GhGA20ox1, showed GA over-production phenotypes with increased endogenous GA levels (especially GA(4)) in fibers and ovules. It also produced significantly more fiber initials per ovule, and fiber lengths was increased compared with the control, which demonstrates that up-regulation of the GhGA20ox1 gene promoted fiber initiation and elongation. Our results suggest that GA 20-oxidase is involved in fiber development by regulating GA levels, and corresponding genes might be employed as target genes for the manipulation of fiber initiation and elongation in cotton.

Characterization and expression of an nsLTPs-like antimicrobial protein gene from motherwort (Leonurus japonicus).

In screening for potent antimicrobial proteins (AMPs) from plant seeds, we had purified a heat-stable AMP, LJAMP2, from the seeds of a medicine herb, motherwort (Leonurus japonicus Houtt). In an in vitro assay, the protein can inhibit the growth of both fungi and bacteria. Then a cDNA encoding LJAMP2 was cloned by the rapid amplification of cDNA ends based on the N-terminal amino acid sequence determined. The deduced amino acid sequences of this cDNA show similarity to plant non-specific lipid transfer proteins. Northern blotting assay revealed that this nsLTP-like gene, designated LJAMP2, was expressed in seeds. Overexpression of LJAMP2 in tobacco enhanced resistance to the fungal pathogen Alternaria alternata and the bacterial pathogen Ralstonia solanacearum, significantly, while no visible alteration in plant growth and development. Our data confirm the antifungal and antibacterial function of LJAMP2 from motherwort seeds and suggest the potential of LJAMP2 in improving disease resistance in plants.

GhDET2, a steroid 5alpha-reductase, plays an important role in cotton fiber cell initiation and elongation.

Cotton (Gossypium hirsutum L.) fibers, one of the most important natural raw materials for textile industry, are highly elongated trichomes from epidermal cells of cotton ovules. DET2, an Arabidopsis steroid 5d-reductase, is considered to catalyze a major rate-limiting in brassinosteroid (BR) biosynthesis. To understand the role of BRs in cotton fiber development, GhDET2, which putatively encodes a steroid 5alpha-reductase by sequence comparison, was cloned from developing fiber cells. In vitro assessment of GhDET2 protein activity confirmed that GhDET2 encodes a functional steroid 5alpha-redutase. High levels of GhDET2 transcript were detected during the fiber initiation stage and the fiber rapid elongation stage. Antisense-mediated suppression of GhDET2 inhibited both fiber initiation and fiber elongation. Similarly, treating cultured ovules with finasteride, a steroid 5alpha-reductase inhibitor, reduced fiber elongation. Inhibition of fiber cell elongation by expression of antisense GhDET2 or the finasteride treatment could be reversed by epibrassinolide, a biologically active BR. Furthermore, seed coat-specific expression of GhDET2 increased fiber number and length. Therefore, GhDET2 and BRs play a crucial role in the initiation and elongation of cotton fiber cells, suggesting that modulation of BR biosynthesis factors may improve fiber quality or yield.

Increased insect virulence in Beauveria bassiana strains overexpressing an engineered chitinase.

Entomopathogenic fungi are currently being used for the control of several insect pests as alternatives or supplements to chemical insecticides. Improvements in virulence and speed of kill can be achieved by understanding the mechanisms of fungal pathogenesis and genetically modifying targeted genes, thus improving the commercial efficacy of these biocontrol agents. Entomopathogenic fungi, such as Beauveria bassiana, penetrate the insect cuticle utilizing a plethora of hydrolytic enzymes, including chitinases, which are important virulence factors. Two chitinases (Bbchit1 and Bbchit2) have previously been characterized in B. bassiana, neither of which possesses chitin-binding domains. Here we report the construction and characterization of several B. bassiana hybrid chitinases where the chitinase Bbchit1 was fused to chitin-binding domains derived from plant, bacterial, or insect sources. A hybrid chitinase containing the chitin-binding domain (BmChBD) from the silkworm Bombyx mori chitinase fused to Bbchit1 showed the greatest ability to bind to chitin compared to other hybrid chitinases. This hybrid chitinase gene (Bbchit1-BmChBD) was then placed under the control of a fungal constitutive promoter (gpd-Bbchit1-BmChBD) and transformed into B. bassiana. Insect bioassays showed a 23% reduction in time to death in the transformant compared to the wild-type fungus. This transformant also showed greater virulence than another construct (gpd-Bbchit1) with the same constitutive promoter but lacking the chitin-binding domain. We utilized a strategy where genetic components of the host insect can be incorporated into the fungal pathogen in order to increase host cuticle penetration ability.

Overexpression of an Arabidopsis magnesium transport gene, AtMGT1, in Nicotiana benthamiana confers Al tolerance.

Aluminium (Al) toxicity is the most important limiting factor for crop production in acid soil environments worldwide. In some plant species, application of magnesium (Mg(2+)) can alleviate Al toxicity. However, it remains unknown whether overexpression of magnesium transport proteins can improve Al tolerance. Here, the role of AtMGT1, a member of the Arabidopsis magnesium transport family involved in Mg(2+) transport, played in Al tolerance in higher plants was investigated. Expression of 35S::AtMGT1 led to various phenotypic alterations in Nicotiana benthamiana plants. Transgenic plants harbouring 35S::AtMGT1 exhibited tolerance to Mg(2+) deficiency. Element assay showed that the contents of Mg, Mn, and Fe in 35S::AtMGT1 plants increased compared with wild-type plants. Root growth experiment revealed that 100 microM AlCl(3) caused a reduction in root elongation by 47% in transgenic lines, whereas root growth in wild-type plants was inhibited completely. Upon Al treatment, representative transgenic lines also showed a much lower callose deposition, an indicator of increased Al tolerance, than wild-type plants. Taken together, the results have demonstrated that overexpression of ATMGT1 encoding a magnesium transport protein can improve tolerance to Al in higher plants.