Morphophysiological alterations in transgenic rice lines expressing PPDK and ME genes from the C4 model Setaria italica.

C4 plants are superior to C3 plants in terms of productivity and limited photorespiration. PPDK (pyruvate orthophosphate dikinase) and NADP-ME (NADP-dependent malic enzyme) are two important photosynthetic C4-specific enzymes present in the mesophyll cells of C4 plants. To evaluate the effect of C4 enzymes in rice, we developed transgenic rice lines by separately introducing Setaria italica PPDK [SiPPDK] and S. italica ME [SiME] gene constructs under the control of the green tissue-specific maize PPDK promoter. Rice plant lines for both constructs were screened using the polymerase chain reaction (PCR), Southern hybridization, and expression analysis. The best transgenic plant lines for each case were selected for physiological and biochemical characterization. The results from qRT-PCR and enzyme activity analysis revealed higher expression and activity of both PPDK and NADP-ME genes compared with the nontransformed and empty-vector-transformed plants. The average photosynthetic efficiency of transgenic plant lines carrying the PPDK and NADP-ME genes increased by 18% and 12%, respectively, and was positively correlated with the increased accumulation of photosynthetic pigment. The decrease in Fv/Fm, increased electron transport rate (ETR), and increased photochemical quenching (qP) compared with nontransformed control plants suggest that transgenic rice plants transferred more absorbed light energy to photochemical reactions than wild-type plants. SiME-transgenic plants displayed reduced leaf malate content and superior performance under water deficit conditions. Interestingly, the transgenic plants showed yield enhancement by exhibiting increased plant height, panicle length, panicle weight and thousand grain weight. Overall, the exogenous foxtail millet C4 gene PPDK enhanced photosynthesis and yield to a greater extent than NADP-ME.

Formation of Proto-Kranz in C3 Rice Induced by Spike-Stalk Injection Method.

Introduction of C4 photosynthetic traits into C3 crops is an important strategy for improving photosynthetic capacity and productivity. Here, we report the research results of a variant line of sorghum-rice (SR) plant with big panicle and high spikelet density by introducing sorghum genome DNA into rice by spike-stalk injection. The whole-genome resequencing showed that a few sorghum genes could be integrated into the rice genome. Gene expression was confirmed for two C4 photosynthetic enzymes containing pyruvate, orthophosphate dikinase and phosphoenolpyruvate carboxykinase. Exogenous sorghum DNA integration induced a series of key traits associated with the C4 pathway called "proto-Kranz" anatomy, including leaf thickness, bundle sheath number and size, and chloroplast size in bundle sheath cells. Significantly, transgenic plants exhibited enhanced photosynthetic capacity resulting from both photosynthetic CO2-concentrating effect and improved energy balance, which led to an increase in carbohydrate levels and productivity. Furthermore, such rice plant exhibited delayed leaf senescence. In summary, this study provides a proof for the feasibility of inducing the transition from C3 leaf anatomy to proto-Kranz by spike-stalk injection to achieve efficient photosynthesis and increase productivity.

RNAi-mediated down-regulation of ITPK-2 enhanced inorganic phosphorus and minerals in the transgenic rice.

Phytic acid or Myo-inositol hexakisphosphate is an essential compound for the rice plants. It remains in the form of phytate, a mixed salt of different mineral cations, in the seeds. The phytate breaks down during germination and provides the inorganic phosphorus and mineral ions to the seedlings. However, humans do not get the benefit of those essential ions from rice consumption due to the absence of phytase in the gut. We envisaged down-regulating ITPK, the gene behind the phytic acid biosynthesis so that its low amount would facilitate a greater amount of free mineral ions in the endosperm. Since there are six homologues of rice ITPK, we studied their expression in seeds. Additionally, we undertook an in-silico analysis of the homologous proteins. Considering the results, we selected ITPK-2 for its RNAi-mediated embryo-specific down-regulation to obtain the low phytate rice. We obtained a 37% reduction of phytic acid content accompanied by a nearly three-fold enhancement of inorganic phosphorus in the transgenic seeds. Additionally, the iron and zinc content increased in polished rice grains compared to the wild type. The results also showed that reduced phytic acid content did not affect the germination potential and seedling growth of the transgenic rice.

Overexpression of a Pak Choi Gene, BcAS2, Causes Leaf Curvature in Arabidopsis thaliana.

The LBD (Lateral Organ Boundaries Domain) family are a new group of plant-specific genes, which encode a class of transcription factors containing conserved Lateral Organization Boundary (LOB) domains, and play an important role in regulating the adaxial-abaxial polarity of plant leaves. In Arabidopsis thaliana, ASYMMETRIC LEAVES 2 (AS2) has a typical LOB domain and is involved in determining the adaxial cell fate. In this study, we isolated the BcAS2 gene from the pak choi cultivar "NHCC001", and analyzed its expression pattern. The results showed that the BcAS2 encoded a protein made up of 202 amino acid residues which were located in the nucleus and cytomembrane. The Yeast two-hybrid system (Y2H) assay indicated that BcAS2 interacts with BcAS1-1 and BcAS1-2 (the homologous genes of AS1 gene in pak choi). In the transgenic Arabidopsis thaliana that overexpressed BcAS2 gene, it presented an abnormal phenotype with a curly shape. Taken together, our findings not only validate the function of BcAS2 in leaf development in Arabidopsis thaliana, but also contribute in unravelling the molecular regulatory mechanism of BcAS2, which fulfills a special role by forming complexes with BcAS1-1/2 in the establishment of the adaxial-abaxial polarity of the lateral organs in pak choi.

Genetics of days to flowering, maturity and plant height in natural and derived forms of Brassica rapa L.

KEY MESSAGE: Genome wide association studies enabled prediction of many candidate genes for flowering, maturity and plant height under differing day-length conditions. Some genes were envisaged only from derived B. rapa. Flowering and plant height are the key life history traits. These are crucial for adaptation and productivity. Current investigations aimed to examine genotypic differences governing days to flowering, maturity and plant height under contrasting day-length conditions; and identify genomic regions governing the observed phenotypic variations. An association panel comprising 195 inbred lines, representing natural (NR) and derived (DR) forms of Brassica rapa (AA; 2n = 20), was evaluated at two sowing dates and two locations, representing different day-length regimes. Derived B. rapa is a unique pre-breeding material extracted from B. juncea (AABB; 2n = 36). Population structure analysis, using DArT genotypes established derived B. rapa as a genetic resource distinct from natural B. rapa. Genome wide association studies facilitated detection of many trait associated SNPs. Chromosomes A03, A05 and A09 harboured majority of these. Functional annotation of the associated SNPs and surrounding genome space(s) helped to predict 43 candidate genes. Many of these were predicted under specific day-length conditions. Important among these were the genes encoding floral meristem identity (SPL3, SPL15, AP3, BAM2), photoperiodic responses (COL2, AGL18, SPT, NF-YC4), gibberellic acid biosynthesis (GA1) and regulation of flowering (EBS). Some of the predicted genes were detected for DR subpanel alone. Genes controlling hormones, auxins and gibberellins appeared important for the regulation of plant height. Many of the significant SNPs were located on chromosomes harbouring previously reported QTLs and candidate genes. The identified loci may be used for marker-assisted selection after due validation.

Probing the role of cell wall feruloylation during maize development by differential expression of an apoplast targeted fungal ferulic acid esterase.

While many aspects of the growth of maize are well understood, the role of cell wall feruloylation particularly during internode elongation has not been firmly established, but results so far indicate that it has significant implications for both biofuel feedstock conversion and for crop yield. The growth of the cell wall is achieved by synthesis, integration and cross-linking between wall polymers. As ferulate oxidative coupling of arabinoxylan side chains constitutes a significant type of cross-link in grass cell walls, it is expected to have a crucial role in plant growth. Making use of plants expressing an apoplast targeted Aspergillus niger FAEA under the control of either a constitutive or an inducible promoter, the role of cell wall feruloylation in maize internode expansion was investigated. Analysis of FAEA expressing plants showed that where FAEA was targeted to the apoplast under a constitutive promoter, plants varied in stature either from semi-dwarf plants with a 40-60% height reduction, to extreme dwarf mutants with over 90% reduction in plant heights compared to controls. Results indicate that disruption of cell wall feruloylation by FAEA occurs before the start of rapid internode expansion is initiated and affects the normal course of internode elongation, resulting in short internodes and dwarfed plants. In contrast, when under the inducible Lm See1 senescence promoter, FAEA activity was found to be low up to the VT stage of development but increased significantly at the VR stage as plants began to senesce, strongly suggesting that normal cell wall feruloylation is required for the process of internode expansion. In addition, with apoplast targeted expression of FAEA under control of the senescence enhanced promoter it was possible to demonstrate decreased cell wall feruloylation without affecting internode expansion or other aspects of plant development.

AgNAC1, a celery transcription factor, related to regulation on lignin biosynthesis and salt tolerance.

The NAC transcription factor participates in various biotic and abiotic stress responses and plays a critical role in plant development. Lignin is a water-insoluble dietary fiber, but it is second only to cellulose in abundance. Celery is the main source of dietary fiber, but its quality and production are limited by various abiotic stresses. Here, AgNAC1 containing the NAM domain was identified from celery. AgNAC1 was found to be a nuclear protein. Transgenic Arabidopsis thaliana plants hosting AgNAC1 have longer root lengths and stomatal axis lengths than the wide type (WT). The evidence from lignin determination and expression levels of lignin-related genes indicated that AgNAC1 plays a vital role in lignin biosynthesis. Furthermore, the results of the physiological characterization and the drought and salt treatments indicate that AgNAC1-overexpressing plants are significantly resistive to salt stress. Under drought and salt treatments, the AgNAC1 transgenic Arabidopsis thaliana plants presented increased superoxide dismutase (SOD) and peroxidase (POD) activities and decreased malondialdehyde (MDA) content and size of stomatal apertures relatively to the WT plants. The AgNAC1 served as a positive regulator in inducing the expression of stress-responsive genes. Overall, the overexpressing AgNAC1 enhanced the plants' resistance to salt stress and played a regulatory role in lignin accumulation.

A CACTA-like transposable element in the upstream region of BnaA9.CYP78A9 acts as an enhancer to increase silique length and seed weight in rapeseed.

Rapeseed (Brassica napus L.) is a model plant for polyploid crop research and the second-leading source of vegetable oil worldwide. Silique length (SL) and seed weight are two important yield-influencing traits in rapeseed. Using map-based cloning, we isolated qSLWA9, which encodes a P450 monooxygenase (BnaA9.CYP78A9) and functions as a positive regulator of SL. The expression level of BnaA9.CYP78A9 in silique valves of the long-silique variety is much higher than that in the regular-silique variety, which results in elongated cells and a prolonged phase of silique elongation. Plants of the long-silique variety and transgenic plants with high expression of BnaA9.CYP78A9 had a higher concentration of auxin in the developing silique; this induced a number of auxin-related genes but no genes in well-known auxin biosynthesis pathways, suggesting that BnaA9.CYP78A9 may influence auxin concentration by affecting auxin metabolism or an unknown auxin biosynthesis pathway. A 3.7-kb CACTA-like transposable element (TE) inserted in the 3.9-kb upstream regulatory sequence of BnaA9.CYP78A9 elevates the expression level, suggesting that the CACTA-like TE acts as an enhancer to stimulate high gene expression and silique elongation. Marker and sequence analysis revealed that the TE in B. napus had recently been introgressed from Brassica rapa by interspecific hybridization. The insertion of the TE is consistently associated with long siliques and large seeds in both B. napus and B. rapa collections. However, the frequency of the CACTA-like TE in rapeseed varieties is still very low, suggesting that this allele has not been widely used in rapeseed breeding programs and would be invaluable for yield improvement in rapeseed breeding.

The DFR locus: A smart landing pad for targeted transgene insertion in tomato.

Targeted insertion of transgenes in plants is still challenging and requires further technical innovation. In the present study, we chose the tomato DFR gene involved in anthocyanin biosynthesis as a landing pad for targeted transgene insertion using CRISPR-Cas9 in a two-step strategy. First, a 1013 bp was deleted in the endogenous DFR gene. Hypocotyls and callus of in vitro regenerated plantlets homozygous for the deletion were green instead of the usual anthocyanin produced purple colour. Next, standard Agrobacterium-mediated transformation was used to target transgene insertion at the DFR landing pad in the dfr deletion line. The single binary vector carried two sgRNAs, a donor template containing two homology arms of 400 bp, the previously deleted DFR sequence, and a NptII expression cassette. Regenerating plantlets were screened for a purple-colour phenotype indicating that DFR function had been restored. Targeted insertions were identified in 1.29% of the transformed explants. Thus, we established an efficient method for selecting HDR-mediated transgene insertion using the CRISPR-Cas9 system in tomato. The visual screen used here facilitates selection of these rare gene targeting events, does not necessitate the systematic PCR screening of all the regenerating material and can be potentially applied to other crops.

Alterations of plant architecture and phase transition by the phytoplasma virulence factor SAP11.

As key mediators linking developmental processes with plant immunity, TCP (TEOSINTE-BRANCHED, CYCLOIDEA, PROLIFERATION FACTOR 1 and 2) transcription factors have been increasingly shown to be targets of pathogenic effectors. We report here that TB/CYC (TEOSINTE-BRANCHED/CYCLOIDEA)-TCPs are destabilized by phytoplasma SAP11 effectors, leading to the proliferation of axillary meristems. Although a high degree of sequence diversity was observed among putative SAP11 effectors identified from evolutionarily distinct clusters of phytoplasmas, these effectors acquired fundamental activity in destabilizing TB/CYC-TCPs. In addition, we demonstrate that miR156/SPLs and miR172/AP2 modules, which represent key regulatory hubs involved in plant phase transition, were modulated by Aster Yellows phytoplasma strain Witches' Broom (AY-WB) protein SAP11. A late-flowering phenotype with significant changes in the expression of flowering-related genes was observed in transgenic Arabidopsis plants expressing SAP11AYWB. These morphological and molecular alterations were correlated with the ability of SAP11 effectors to destabilize CIN (CINCINNATA)-TCPs. Although not all putative SAP11 effectors display broad-spectrum activities in modulating morphological and physiological changes in host plants, they serve as core virulence factors responsible for the witches' broom symptom caused by phytoplasmas.

Functional conservation of Arabidopsis LNG1 in tobacco relating to leaf shape change by increasing longitudinal cell elongation by overexpression.

The LONGIFOLIA1 (LNG1) gene of Arabidopsis regulates leaf shape by polar cell elongation independent of ROTUNDAFOLIA3 (ROT3). To expand our knowledge on the function of this gens in plant systems, Arabidopsis LNG1 (AtLNG1) was introduced both sense and antisense orientation under the control of 35S CaMV promoter into tobacco plants that lack AtLNG1 homolog. Resulting transgenic tobacco plants were analyzed by their phenotype, anatomy and transcript levels. AtLNG1-overexpressing tobacco lines showed increase in the leaf petiole and leaf blade compared with wild type tobacco line. The overexpressors also showed elongated palisade cells as well as epidermal cells in the leaf length direction, but no increase in cell number. Ectopic expression of AtLNG1 in tobacco plants also increased the expression of cell wall modification-related genes, such as NT_XYLOGLUCAN ENDOTRANSGLUCOSYLASE/HYDROLASE9 (NT_XTH9), NT_XTH15 and NT_XTH33, indicating that these genes appear to be target of AtLNG1. As results of molecular and cellular examination, AtLNG1 seemed to have a conserved functional role in shaping leaf morphology in both Arabidopsis and tobacco.

Iris lactea var. chinensis (Fisch.) cysteine-rich gene llCDT1 enhances cadmium tolerance in yeast cells and Arabidopsis thaliana.

IlCDT1, a cysteine-rich protein, was isolated from Iris lactea var. chinensis (Fisch.) (I. lactea var. chinensis). Its transcription was up-regulated by the exogenous application of Cd. The truncated IlCDT1 (25-54) containing 14 Cys residues confers Cd tolerance to yeast as the intact IlCDT1, indicating that Cys residues are required for Cd tolerance presumably by chelating Cd. When the gene was constitutively expressed in A. thaliana, root length of transgenic lines was longer than that of wild-type under 100 µM or 200 µM Cd stress. However, Cd absorption in wild-type was more than in two trangenic lines under 100 µM Cd exposure. IlCDT1 may directly bind Cd, through chelating Cd and avoiding the Cd uptake into the cells. Together, IlCDT1 may be a promising gene for the Cd tolerance improvement. SUMMARY: Cysteine-rich gene llCDT1 enhances cadmium tolerance in yeast cells and Arabidopsis thaliana.

OsPRX2 contributes to stomatal closure and improves potassium deficiency tolerance in rice.

Peroxiredoxins (Prxs) which are thiol-based peroxidases have been implicated in the toxic reduction and intracellular concentration regulation of hydrogen peroxide. In Arabidopsis thaliana At2-CysPrxB (At5g06290) has been demonstrated to be essential in maintaining the water-water cycle for proper H2O2 scavenging. Although the mechanisms of 2-Cys Prxs have been extensively studied in Arabidopsis thaliana, the function of 2-Cys Prxs in rice is unclear. In this study, a rice homologue gene of At2-CysPrxB, OsPRX2 was investigated aiming to characterize the effect of 2-Cys Prxs on the K+-deficiency tolerance in rice. We found that OsPRX2 was localized in the chloroplast. Overexpressed OsPRX2 causes the stomatal closing and K+-deficiency tolerance increasing, while knockout of OsPRX2 lead to serious defects in leaves phenotype and the stomatal opening under the K+-deficiency tolerance. Detection of K+ accumulation, antioxidant activity of transgenic plants under the starvation of potassium, further confirmed that OsPRX2 is a potential target for engineering plants with improved potassium deficiency tolerance.

Differential rubisco content and photosynthetic efficiency of rol gene integrated Vinca minor transgenic plant: Correlating factors associated with morpho-anatomical changes, gene expression and alkaloid productivity.

Transgenic plants obtained from a hairy root line (PVG) of Vinca minor were characterized in relation to terpenoid indole alkaloids (TIAs) pathway gene expression and vincamine production. The hairy roots formed callus with green nodular protuberances when transferred onto agar-gelled MS medium containing 3.0mg/l zeatin. These meristematic zones developed into shoot buds on medium with 1.0mg/l 2, 4-dichlorophenoxyacetic acid and 40mg/l ascorbic acid. These shoot buds subsequently formed rooted plants when shifted onto a hormone-free MS medium with 6% sucrose. Transgenic nature of the plants was confirmed by the presence of rol genes of the Ri plasmid in them. The transgenic plants (TP) had elongated internodes and a highly proliferating root system. During glass house cultivation TP consistently exhibited slower growth rate, low chlorophyll content (1.02±0.08mg/gm fr. wt.), reduced carbon exchange rate (2.67±0.16µmolm-2s-1), less transpiration rate (2.30±0.20mmolm-2 s-1) and poor stomatal conductance (2.21±0.04mmolm-2 s-1) when compared with non-transgenic population. The activity of rubisco enzyme in the leaves of TP was nearly two folds less in comparison to non-transgenic controls (1.80milliunitsml-1mgprotein-1 against 3.61milliunits ml-1mgprotein-1, respectively). Anatomically, the TP had a distinct tetarch arrangement of vascular bundles in their stem and roots against a typical ployarched pattern in the non-transgenic plants. Significantly, the transgenic plants accumulated 35% higher amount of total TIAs (3.10±0.21% dry wt.) along with a 0.03% dry wt. content of its vasodilatory and nootropic alkaloid vincamine in their leaves. Higher productivity of alkaloids in TP was corroborated with more than four (RQ=4.60±0.30) and five (RQ=5.20±0.70) times over-expression of TIAs pathway genes tryptophan decarboxylase (TDC) and strictosidine synthase (STR) that are responsible for pushing the metabolic flux towards TIAs synthesis in this medicinal herb.

Overexpression of SlPRE2, an atypical bHLH transcription factor, affects plant morphology and fruit pigment accumulation in tomato.

The basic helix-loop-helix (bHLH) proteins are a large family of transcription factors that control various developmental processes in eukaryotes, but the biological roles of most bHLH proteins are not very clear, especially in tomato. In this study, a PRE-like atypical bHLH gene was isolated and designated as SlPRE2 in tomato. SlPRE2 was highly expressed in immature-green fruits, moderately in young leaves, flowers, and mature-green fruits. To further research the function of SlPRE2, a 35 S:PRE2 binary vector was constructed and transformed into wild type tomato. The transgenic plants showed increased leaf angle and stem internode length, rolling leaves with decreased chlorophyll content. The water loss rate of detached leaves was increased in young transgenic lines but depressed in mature leaves. Besides, overexpression of SlPRE2 promoted morphogenesis in seedling development, producing light-green unripening fruits and yellowing ripen fruits with reduced chlorophyll and carotenoid accumulation in pericarps, respectively. Quantitative RT-PCR analysis showed that expression of the chlorophyll related genes, such as GOLDEN 2-LIKE and RbcS, were decreased in unripening 35 S:PRE2 fruit, and carotenoid biosynthesis-related genes PHYTOENE SYNTHASE1A and ζ-CAROTENE DESATURASE in ripening fruit were also down-regulated. These results suggest that SlPRE2 affects plant morphology and is a negative regulator of fruit pigment accumulation.

Ectopic expression of Arabidopsis FD and FD PARALOGUE in rice results in dwarfism with size reduction of spikelets.

Key flowering genes, FD and FD PARALOGUE (FDP) encoding bZIP transcription factors that interact with a FLOWERING LOCUS T (FT) in Arabidopsis were ectopically expressed in rice since we found AtFD and AtFDP also interact with HEADING DATE 3a (Hd3a) and RICE FLOWERING LOCUS T 1 (RFT1). Transgenic rice plants overexpressing AtFD and AtFDP caused reduction in plant height and spikelet size with decreased expression of genes involved in cell elongation without significant flowering time alteration in spite of increased expression of OsMADS14 and OsMADS15, rice homologues of APETALA1 (AP1) in the leaves. Simultaneous overexpression of AtFD and AtFDP enhanced phenotypes seen with overexpression of either single gene while transgenic rice plants expressing AtFD or AtFDP under the control of phloem-specific Hd3a promoter were indistinguishable from wild-type rice. Candidate genes responsible for the phenotypes were identified by comparison of microarray hybridization and their expression pattern was also examined in WT and transgenic rice plants. It has so far not been reported that AtFD and AtFDP affect cell elongation in plants, and our findings provide novel insight into the possible roles of AtFD and AtFDP in the mesophyll cells of plants, and potential genetic tools for manipulation of crop architecture.

HvDep1 Is a Positive Regulator of Culm Elongation and Grain Size in Barley and Impacts Yield in an Environment-Dependent Manner.

Heterotrimeric G proteins are intracellular membrane-attached signal transducers involved in various cellular processes in both plants and animals. They consist of three subunits denoted as α, ß and γ. The γ-subunits of the so-called AGG3 type, which comprise a transmembrane domain, are exclusively found in plants. In model species, these proteins have been shown to participate in the control of plant height, branching and seed size and could therefore impact the harvestable yield of various crop plants. Whether AGG3-type γ-subunits influence yield in temperate cereals like barley and wheat remains unknown. Using a transgenic complementation approach, we show here that the Scottish malting barley cultivar (cv.) Golden Promise carries a loss-of-function mutation in HvDep1, an AGG3-type subunit encoding gene that positively regulates culm elongation and seed size in barley. Somewhat intriguingly, agronomic field data collected over a 12-year period reveals that the HvDep1 loss-of-function mutation in cv. Golden Promise has the potential to confer either a significant increase or decrease in harvestable yield depending on the environment. Our results confirm the role of AGG3-type subunit-encoding genes in shaping plant architecture, but interestingly also indicate that the impact HvDep1 has on yield in barley is both genotypically and environmentally sensitive. This may explain why widespread exploitation of variation in AGG3-type subunit-encoding genes has not occurred in temperate cereals while in rice the DEP1 locus is widely exploited to improve harvestable yield.

Overexpression of the Transcription Factors GmSHN1 and GmSHN9 Differentially Regulates Wax and Cutin Biosynthesis, Alters Cuticle Properties, and Changes Leaf Phenotypes in Arabidopsis.

SHINE (SHN/WIN) clade proteins, transcription factors of the plant-specific APETALA 2/ethylene-responsive element binding factor (AP2/ERF) family, have been proven to be involved in wax and cutin biosynthesis. Glycine max is an important economic crop, but its molecular mechanism of wax biosynthesis is rarely characterized. In this study, 10 homologs of Arabidopsis SHN genes were identified from soybean. These homologs were different in gene structures and organ expression patterns. Constitutive expression of each of the soybean SHN genes in Arabidopsis led to different leaf phenotypes, as well as different levels of glossiness on leaf surfaces. Overexpression of GmSHN1 and GmSHN9 in Arabidopsis exhibited 7.8-fold and 9.9-fold up-regulation of leaf cuticle wax productions, respectively. C31 and C29 alkanes contributed most to the increased wax contents. Total cutin contents of leaves were increased 11.4-fold in GmSHN1 overexpressors and 5.7-fold in GmSHN9 overexpressors, mainly through increasing C16:0 di-OH and dioic acids. GmSHN1 and GmSHN9 also altered leaf cuticle membrane ultrastructure and increased water loss rate in transgenic Arabidopsis plants. Transcript levels of many wax and cutin biosynthesis and leaf development related genes were altered in GmSHN1 and GmSHN9 overexpressors. Overall, these results suggest that GmSHN1 and GmSHN9 may differentially regulate the leaf development process as well as wax and cutin biosynthesis.

[Morphological Features of the Transgenic Tobacco Plant Shoot Expressing the 3-Hydroxy-3-Methylglutagyl-CoA Reductase (HMG1) Gene in the Direct and Reverse Orientations Towards the Promoter].

3-Hydroxy-3-methylglutaryl-CoA reductase (HMG1) catalyzes the formation of mevalonic acid, the key intermediate of the cytosolic isoprenoid synthesis pathway. The parameters of stem and leaf growth were studied in the transgenic tobacco plants that express the HMG1 gene in both sense and antisense orientations towards the constitutive promoter. The transgenic plant height did not significantly differ from that of the control plants, though the plants carrying the sense copy of the HMG1 gene were considerably taller than plants that carried the antisense gene copy. Plants carrying an extra copy of the HMG1 gene were also characterized by increased leaf area. The number of mesophyll cells calculated per square unit of transgenic plants leaves was smaller than in the control plant leaves, though their volume was not considerably changed in any of the variants, suggesting changes in the cell packing density in leaves.

Expression of kenaf mitochondrial chimeric genes HM184 causes male sterility in transgenic tobacco plants.

Chimeric genes resulting from the rearrangement of a mitochondrial genome were generally thought to be a causal factor in the occurrence of cytoplasmic male sterility (CMS). In the study, earlier we reported that identifying a 47 bp deletion at 3'- flanking of atp9 that was linked to male sterile cytoplasm in kenaf. The truncated fragment was fused with atp9, a mitochondrial transit signal (MTS) and/or GFP, comprised two chimeric genes MTS-HM184-GFP and MTS-HM184. The plant expression vector pBI121 containing chimeric genes were then introduced to tobacco plants by Agrobacterium-mediated T-DNA transformation. The result showed that certain transgenic plants were male sterility or semi-sterility, while some were not. The expression analysis further demonstrated that higher level of expression were showed in the sterility plants, while no expression or less expression in fertility plants, the levels of expression of semi-sterility were in between. And the sterile plant (containing MTS-HM184-GFP) had abnormal anther produced malformed/shriveled pollen grains stained negative that failed to germinate (0%), the corresponding fruits was shrunken, the semi-sterile plants having normal anther shape produced about 10-50% normal pollen grains, the corresponding fruits were not full, and the germination rate was 58%. Meanwhile these transgenic plants which altered on fertility were further analyzed in phenotype. As a result, the metamorphosis leaves were observed in the seedling stage, the plant height of transgenic plants was shorter than wild type. The growth duration of transgenic tobacco was delayed 30-45 days compared to the wild type. The copy numbers of target genes of transgenic tobacco were analyzed using the real-time quantitative method. The results showed that these transgenic plants targeting-expression in mitochondrial containing MTS-HM184-GFP had 1 copy and 2 copies, the other two plants containing MTS-HM184 both had 3 copies, but 0 copy in wild type. In summary, the two manual chimeric genes might be related to male sterility in kenaf.