RNA Interference Transgenic Lines Research Articles

Functional analysis of polyphenol oxidase 1 gene in common wheat.

Polyphenol oxidase (PPO) activity is a major cause of the undesirable brown color of wheat-based products. Ppo1, a major gene for PPO activity, was cloned based on sequence homology in previous studies; however, its function and regulation mechanism remain unclear. In this study, the function and genetic regulation of Ppo1 were analyzed using RNA interference (RNAi) and Targeting Induced Local Lesions IN Genomes (TILLING) technology, and superior mutants were identified. Compared with the control, the level of Ppo1 transcript in RNAi transgenic lines was drastically decreased by 15.5%-60.9% during grain development, and PPO activity was significantly reduced by 12.9%-20.4%, confirming the role of Ppo1 in PPO activity. Thirty-two Ppo1 mutants were identified in the ethyl methanesulfonate (EMS)-mutagenized population, including eight missense mutations, 16 synonymous mutations, and eight intron mutations. The expression of Ppo1 was reduced significantly by 6.7%-37.1% and 10.1%-54.4% in mutants M092141 (G311S) and M091098 (G299R), respectively, in which PPO activity was decreased by 29.7% and 28.8%, respectively, indicating that mutation sites of two mutants have important effects on PPO1 function. Sequence and structure analysis revealed that the two sites were highly conserved among 74 plant species, where the frequency of glycine was 94.6% and 100%, respectively, and adjacent to the entrance of the hydrophobic pocket of the active site. The M092141 and M091098 mutants can be used as important germplasms to develop wheat cultivars with low grain PPO activity. This study provided important insights into the molecular mechanism of Ppo1 and the genetic improvement of wheat PPO activity.

PeCLH2 Gene Positively Regulate Salt Tolerance in Transgenic Populus alba × Populus glandulosa.

Salt is an important environmental stress factor, which seriously affects the growth, development and distribution of plants. Chlorophyllase plays an important role in stress response. Nevertheless, little is known about the physiological and molecular mechanism of chlorophyll (Chlase, CLH) genes in plants. We cloned PeCLH2 from Populus euphratica and found that PeCLH2 was differentially expressed in different tissues, especially in the leaves of P. euphratica. To further study the role of PeCLH2 in salt tolerance, PeCLH2 overexpression and RNA interference transgenic lines were established in Populus alba × Populus glandulosa and used for salt stress treatment and physiologic indexes studies. Overexpressing lines significantly improved tolerance to salt treatment and reduced reactive oxygen species production. RNA interference lines showed the opposite. Transcriptome analysis was performed on leaves of control and transgenic lines under normal growth conditions and salt stress to predict genes regulated during salt stress. This provides a basis for elucidating the molecular regulation mechanism of PeCLH2 in response to salt stress and improving the tolerance of poplar under salt stress.

Distinguishing resistances of transgenic sugarcane generated from RNA interference and pathogen‐derived resistance approaches to combating sugarcane mosaic virus

Sugarcane mosaic virus (SCMV) is a causative agent that reduces growth and productivity in sugarcane. Pathogen‐derived resistance (PDR) and RNA interference (RNAi) are the most common approaches to generating resis‐ tance against plant viruses. Two types of transgenic sugarcane have been obtained by PDR and RNAi methods using a gene‐encoding coat protein (CP) of SCMV (SCMVCp). This research aimed to distinguish resistance of the two transgenic sugarcanes in combating SCMV through artificial viral inoculation. The experiment was conducted using transgenic sugar‐ cane lines validated by PCR analysis. Insertion of gene‐encoding CP in the transgenic lines was confirmed by amplification of 702 bp of DNA fragment of SCMVCp. After viral inoculation, mosaic symptoms appeared earlier, at 21 days post inoculation (dpi) in PDR transgenic lines, but was at 26 dpi in RNAi transgenic lines. Symptom observation showed that 77.8% and 50% of the inoculated plants developed mosaic symptoms in PDR and RNAi transgenic lines, respectively. RT‐PCR analysis revealed that the nuclear inclusion protein b (Nib) gene of SCMV was amplified in the symptomatic leaves in plants classified as susceptible lines. Immunoblot analysis confirmed presence of viral CP with a molecular size of 37 kDa in the susceptible lines. Collectively, these results indicated that the RNAi approach targeting the gene for CP effectively produces more resistance against the SCMV infection in transgenic sugarcane compared to the PDR approach.

Three Paralogous R2R3-MYB Genes Contribute to Delphinidin-Related Anthocyanins Synthesis in Petunia hybrida

Three ANTHOCYANIN SYNTHESIS REGULATOR genes (ASR1–3) that encode R2R3-MYB transcription factors were identified recently from Petunia hybrida. In this study, we conducted additional experiments to characterize their specific function in the regulation of anthocyanin synthesis. The ASR1–3 proteins were localized in the nucleus. Analysis of their regulatory function by transient expression in the petunia corolla and yeast two-hybrid assays showed that the residues Arg at position 51 and Ala at position 102 in the N-terminal domain were essential for the regulatory function of the proteins, and the conserved domain at the C-terminal end was important for activation of the protein. RNA sequencing of overexpression (OE) and RNA-interference transgenic lines confirmed that the ASR proteins specifically induce structural genes of anthocyanin synthesis, including the early biosynthesis genes CHSj, F3H, and F3′5′H-1, the late biosynthesis genes DFR, ANS, RT, MT, AT, and GT, and the anthocyanin-related glutathione S-transferase gene AN9. In addition, a member of the detoxifying efflux carrier family, a DTX35-like gene, was upregulated by ASRs. Determination of anthocyanin/anthocyanidin contents in ASR1-OE petunia lines revealed that ASR1 especially induced the flavonoid 3′,5′-hydroxylase, which was consistent with the abundance of dihydromyricetins and delphinidins, but not cyanidins. The ASR genes were induced under high-intensity light and upregulated the expression of MYBx and MYB27, thereby providing feedback repression of anthocyanin synthesis. An updated model is presented outlining the mechanisms underlying anthocyanin synthesis in petunia.

Silencing of the Ornithine Decarboxylase Gene of Fusarium oxysporum f. sp. lycopersici by Host-Induced RNAi Confers Resistance to Fusarium Wilt in Tomato

Fusarium wilt caused by Fusarium oxysporum f. sp. lycopersici, a hemibiotrophic filamentous fungal pathogen is one of the important diseases of tomato. Recently, RNA interference (RNAi) has emerged as a novel alternative strategy for the control of plant diseases caused by fungal pathogens. Therefore, we tested the potential of RNAi for the control of Fusarium wilt in tomato by targeting a key polyamine (PA) biosynthesis gene, ornithine decarboxylase (ODC) of the pathogen as PAs (putrescine, spermidine, and spermine) are absolutely essential for normal growth and development. The target fungal ODC gene fragment was cloned in a hairpin RNA construct, which was used to develop several transgenic tomato plants. These RNAi transgenic lines expressed the intended small interfering RNAs (siRNAs) and exhibited moderate to high resistance when challenged with the spores of F. oxysporum. Interestingly, the silencing effect was confined only to the fungal pathogen and had no influence on the plant ODC gene expression, polyamine levels, and morphology. These results confirm that the ODC gene is vital for fungal growth and is a suitable target for disease control through host-induced gene silencing (HIGS) and also implicates the transfer of dsRNA/siRNAs from host plant cells to the fungal cells. To further validate the uptake of plant-derived siRNAs by fungal cells in a visual manner, silencing of the GFP transgene was observed in F. oxysporum GFP transformants upon infecting the dsGFP-expressing transgenic tomato plants. These experiments demonstrate the applicability of RNAi-based approach for crop protection from the filamentous fungal pathogens.

Silencing of the MADS-Box Gene SlMADS83 Enhances Adventitious Root Formation in Tomato Plants

Adventitious roots (ARs) are important for the growth of plants and the improvement in their stress resistance and survival capacity. Although many genes have been confirmed to be involved in adventitious root (AR) formation in Arabidopsis and tomato plants, MADS-box genes have rarely been mentioned. Here, we isolated a MADS-box gene named SlMADS83, which may negatively regulate AR formation in tomato plants, as the number of the ARs formed in the transgenic lines in which the SlMADS83 gene was silenced by RNA interference (RNAi) was increased. The above phenotype was further confirmed by the analysis of the macroscopic, anatomical, and molecular features and related statistical data. Previous Studies have proven that auxin can stimulate early AR primordium initiation. Interestingly, in the RNAi transgenic lines, the concentration of auxin in the hypocotyl base was increased, resulting in early induction of AR primordia initiation, promoting the formation of ARs. Briefly, SlMADS83 may play an important role in AR formation.

Molecular cloning, characterization, and promoter analysis of the isochorismate synthase (AaICS1) gene from Artemisia annua.

Isochorismate synthase (ICS) is a crucial enzyme in the salicylic acid (SA) synthesis pathway. The full-length complementary DNA (cDNA) sequence of the ICS gene was isolated from Artemisia annua L. The gene, named AaICS1, contained a 1710-bp open reading frame, which encoded a protein with 570 amino acids. Bioinformatics and comparative study revealed that the polypeptide protein of AaICS1 had high homology with ICSs from other plant species. Southern blot analysis suggested that AaICS1 might be a single-copy gene. Analysis of the 1470-bp promoter of AaICS1 identified distinct cis-acting regulatory elements, including TC-rich repeats, MYB binding site (MBS), and TCA-elements. An analysis of AaICS1 transcript levels in multifarious tissues of A. annua using quantitative real-time polymerase chain reaction (qRT-PCR) showed that old leaves had the highest transcription levels. AaICS1 was up-regulated under wounding, drought, salinity, and SA treatments. This was corroborated by the presence of the predicted cis-acting elements in the promoter region of AaICS1. Overexpressing transgenic plants and RNA interference transgenic lines of AaICS1 were generated and their expression was compared. High-performance liquid chromatography (HPLC) results from leaf tissue of transgenic A. annua showed an increase in artemisinin content in the overexpressing plants. These results confirm that AaICS1 is involved in the isochorismate pathway.

Mitochondrial ABC Transporter ATM3 Is Essential for Cytosolic Iron-Sulfur Cluster Assembly.

The mitochondrial ATP-binding cassette transporter ATM3 has been studied in Arabidopsis. Its function, however, is poorly understood in other model plant species. This study reports that the ATM3 is required for cytosolic iron-sulfur cluster assembly and is essential for meristem maintenance in rice (Oryza sativa). The loss of function of OsATM3 is lethal in rice at the four-leaf stage. In the osatm3 T-DNA insertion mutant, the fourth leaf fails to develop and the lateral roots are short. Cytosolic iron-sulfur protein activities were significantly reduced in both osatm3 and RNA interference transgenic lines. The expression profiles of many iron metabolism genes were altered in the osatm3 and RNA interference lines. Glutathione metabolism was impaired and reactive oxygen species, particularly superoxide, accumulated in osatm3 Promoter-β-glucuronidase staining of the transgenic line indicated that OsATM3 is highly expressed in lateral root primordia, root tip meristem zones, and shoot apical meristem regions. The average cell size was significantly greater in osatm3 than in the wild type. Massive cell death occurred in the osatm3 root tip meristem zone. Quantitative RT-PCR revealed transcriptional reprogramming of the genes in the osatm3 and RNAi lines involved in DNA repair and cell cycle arrest. Our results suggest that the mitochondrial ATM3 is essential for iron homeostasis in rice.

AtPDCD5 Plays a Role in Programmed Cell Death after UV-B Exposure in Arabidopsis

DNA damage responses have evolved to sense and react to DNA damage; the induction of DNA repair mechanisms can lead to genomic restoration or, if the damaged DNA cannot be adequately repaired, to the execution of a cell death program. In this work, we investigated the role of an Arabidopsis (Arabidopsis thaliana) protein, AtPDCD5, which is highly similar to the human PDCD5 protein; it is induced by ultraviolet (UV)-B radiation and participates in programmed cell death in the UV-B DNA damage response. Transgenic plants expressing AtPDCD5 fused to GREEN FLUORESCENT PROTEIN indicate that AtPDCD5 is localized both in the nucleus and the cytosol. By use of pdcd5 mutants, we here demonstrate that these plants have an altered antioxidant metabolism and accumulate higher levels of DNA damage after UV-B exposure, similar to levels in ham1ham2 RNA interference transgenic lines with decreased expression of acetyltransferases from the MYST family. By coimmunoprecipitation and pull-down assays, we provide evidence that AtPDCD5 interacts with HAM proteins, suggesting that both proteins participate in the same pathway of DNA damage responses. Plants overexpressing AtPDCD5 show less DNA damage but more cell death in root tips upon UV-B exposure. Finally, we here show that AtPDCD5 also participates in age-induced programmed cell death. Together, the data presented here demonstrate that AtPDCD5 plays an important role during DNA damage responses induced by UV-B radiation in Arabidopsis and also participates in programmed cell death programs.

Functional Characterization of Cucumis metuliferus Proteinase Inhibitor Gene (CmSPI) in Potyviruses Resistance.

Proteinase inhibitors are ubiquitous proteins that block the active center or interact allosterically with proteinases and are involved in plant physiological processes and defense responses to biotic and abiotic stresses. The CmSPI gene identified from Cucumis metuliferus encodes a serine type PI (8 kDa) that belongs to potato I type family. To evaluate the effect of silencing CmSPI gene on Papaya ringspot virus resistance, RNA interference (RNAi) with an inter-space hairpin RNA (ihpRNA) construct was introduced into a PRSV-resistant C. metuliferus line. CmSPI was down-regulated in CmSPI RNAi transgenic lines in which synchronously PRSV symptoms were evident at 21 day post inoculation. Alternatively, heterogeneous expression of CmSPI in Nicotiana benthamiana was also conducted and showed that CmSPI can provide resistance to Potato virus Y, another member of Potyvirus, in transgenic N. benthamiana lines. This study demonstrated that CmSPI plays an important role in resistant function against potyviruses in C. metuliferus and N. benthamiana.

Synthetic gene complementation to determine off-target silencing.

RNA interference (RNAi) is a conserved mechanism in a wide range of eukaryotes. Introduction of synthetic dsRNA could specifically target suppression of a gene or could result in off-target silencing of another gene due to sequence similarity. To verify if the observed phenotype in an RNAi transgenic line is due to silencing of a specific gene or if it is due to another nontarget gene, a synthetic gene complementation approach could be used. Synthetic gene complementation described in this method uses the technology of synthesizing a variant of a native gene (used in RNAi silencing) to maximize the difference in DNA sequences while coding for the exact same amino acids as the original native gene. This is achieved through the use of alternate codons. The new variant gene is expressed in the original RNAi transgenic lines and analyzed for complementation of the RNAi phenotype. Complementation of the RNAi-induced phenotype will indicate gene-specific silencing and not off-target silencing.

Disruption of prefoldin-2 protein synthesis in root-knot nematodes via host-mediated gene silencing efficiently reduces nematode numbers and thus protects plants.

The aim of this study is to demonstrate the feasibility of down-regulating endogeneous prefoldin-2 root-knot nematode transcripts by expressing dsRNA with sequence identity to the nematode gene in tobacco roots under the influence of strong Arabidopsis ubiquitin (UBQ1) promoter. Root-knot nematodes (RKNs) are sedentary endoparasites infecting a wide range of plant species. They parasitise the root system, thereby disrupting water and nutrient uptake and causing major reductions in crop yields. The most reliable means of controlling RKNs is via the use of soil fumigants such as methyl bromide. With the emergence of RNA interference (RNAi) technology, which permits host-mediated nematode gene silencing, a new strategy to control plant pathogens has become available. In the present study, we investigated host-induced RNAi gene silencing of prefoldin-2 in transgenic Nicotiana benthamiana. Reductions in prefoldin-2 mRNA transcript levels were observed when nematodes were soaked in a dsRNA solution in vitro. Furthermore, nematode reproduction was suppressed in RNAi transgenic lines, as evident by reductions in the numbers of root knots (by 34-60% in independent RNAi lines) and egg masses (by 33-58%). Endogenous expression of prefoldin-2, analysed via real-time polymerase chain reaction and Western blotting, revealed that the gene was strongly expressed in the pre-parasitic J2 stage. Our observations demonstrate the relevance and potential importance of targeting the prefoldin gene during the nematode life cycle. The work also suggests that further improvements in silencing efficiency in economically important crops can be accomplished using RNAi directed against plant-parasitic nematodes.

The rice (Oryza sativa L.) LESION MIMIC RESEMBLING, which encodes an AAA-type ATPase, is implicated in defense response.

Lesion mimic mutants (LMMs) provide a useful tool to study defense-related programmed cell death (PCD) in plants. Although a number of LMMs have been identified in multiple species, most of the candidate genes are yet to be isolated. Here, we report the identification and characterization of a novel rice (Oryza sativa L.) lesion mimic resembling (lmr) mutant, and cloning of the corresponding LMR gene. The LMR locus was initially delineated to 1.2Mb region on chromosome 6, which was further narrowed down to 155-kb using insertions/deletions (INDELs) and cleavage amplified polymorphic sequence markers developed in this study. We sequenced the open reading frames predicted within the candidate genomic region, and identified a G-A base substitution causing a premature translation termination in a gene that encodes an ATPase associated with various cellular activities type (AAA-type) protein. RNA interference transgenic lines with reduced LMR transcripts exhibited the lesion mimic phenotype similar to that of lmr plants. Furthermore, expression of the wild-type LMR in the mutant background complemented the lesion phenotype, confirming that the mutation identified in LMR is responsible for the mutant phenotype. The pathogenesis-related (PR) genes PBZ1 and PR1 were induced in lmr, which also showed enhanced resistance to rice blast (Magnaporthe oryzae) and bacterial blight (Xanthomonas oryzae pv. oryzae), suggesting LMR is a negative regulator of cell death in rice. The identification of lmr and cloning of the corresponding LMR gene provide an additional resource for the study of PCD in plants.

Tudor-SN, a component of stress granules, regulates growth under salt stress by modulating GA20ox3 mRNA levels in Arabidopsis.

The Tudor-SN protein (TSN) is universally expressed and highly conserved in eukaryotes. In Arabidopsis, TSN is reportedly involved in stress adaptation, but the mechanism involved in this adaptation is not understood. Here, we provide evidence that TSN regulates the mRNA levels of GA20ox3, a key enzyme for gibberellin (GA) biosynthesis. The levels of GA20ox3 transcripts decreased in TSN1/TSN2 RNA interference (RNAi) transgenic lines and increased in TSN1 over-expression (OE) transgenic lines. The TSN1 OE lines displayed phenotypes that may be attributed to the overproduction of GA. No obvious defects were observed in the RNAi transgenic lines under normal conditions, but under salt stress conditions these lines displayed slower growth than wild-type (WT) plants. Two mutants of GA20ox3, ga20ox3-1 and -2, also showed slower growth under stress than WT plants. Moreover, a higher accumulation of GA20ox3 transcripts was observed under salt stress. The results of a western blot analysis indicated that higher levels of TSN1 accumulated after salt treatment than under normal conditions. Subcellular localization studies showed that TSN1 was uniformly distributed in the cytoplasm under normal conditions but accumulated in small granules and co-localized with RBP47, a marker protein for stress granules (SGs), in response to salt stress. The results of RNA immunoprecipitation experiments indicated that TSN1 bound GA20ox3 mRNA in vivo. On the basis of these findings, we conclude that TSN is a novel component of plant SGs that regulates growth under salt stress by modulating levels of GA20ox3 mRNA.

Downregulation of Glucan Synthase-Like (TaGSL) genes in wheat leads to inhibition of transgenic plant regeneration

RNA interference (RNAi) cassettes for gene silencing in plants consist of an inverted repeat sequence of the targeted gene and a spacer region separating sense and antisense fragments. The sequences are generally placed under the control of a strong promoter and terminator and produce hairpin RNA structures that generate small interfering RNAs (siRNAs). These siRNAs destroy the mRNA of the matching gene in the cytoplasm. The number and efficiency of siRNAs depend upon the sequence length of inverted repeats and the region in the gene. In the present study, our objective was to downregulate three members of the Glucan Synthase-Like family in wheat. Initially, a 230-nucleotide fragment was cloned under the maize ubiquitin-1 promoter and nos terminator in both sense and antisense directions, and separated by a spacer including the Escherichia coli uidA (GUS) gene. RNAi constructs were prepared for TaGSL8 and TaGSL10. Biolistic delivery of RNAi constructs for TaGSL8 and TaGSL10 into immature zygotic embryos (IZEs) of wheat did not yield any transgenic plants. The experiment was repeated and comparisons were made with a control bar gene construct. IZEs bombarded with a bar selection cassette alone generated healthy calli on selection medium and transgenic plants were recovered. IZEs co-bombarded with the combination of RNAi and bar gene constructs produced only unhealthy calli when cultured on selection medium and no transgenic plants were recovered. This indicated that the RNAi constructs with 230-bp long inverted repeats of TaGSL8 or TaGSL10 were inhibiting regeneration. In the second experiment, the length of the inverted repeats was reduced to 150 and 122 nucleotides for TaGSL3 and TaGSL8, respectively. Two independent transgenic plants were recovered for each of the TaGSL3 and TaGSL8 target genes. These transgenic plants showed transcription of the introduced RNAi constructs, and the transcript levels of the corresponding endogenous genes were reduced. RNAi transgenic lines of TaGSL3 and TaGSL8 showed slightly reduced resistance against Fusarium graminearum compared to nontransgenic control plants.

LeMYC2 acts as a negative regulator of blue light mediated photomorphogenic growth, and promotes the growth of adult tomato plants.

BackgroundArabidopsis ZBF1/MYC2bHLH transcription factor is a repressor of photomorphogenesis, and acts as a point of cross talk in light, abscisic acid (ABA) and jasmonic acid (JA) signaling pathways. MYC2 also functions as a positive regulator of lateral root development and flowering time under long day conditions. However, the function of MYC2 in growth and development remains unknown in crop plants.ResultsHere, we report the functional analyses of LeMYC2 in tomato (Lycopersicon esculentum). The amino acid sequence of LeMYC2 showed extensive homology with Arabidopsis MYC2, containing the conserved bHLH domain. To study the function of LeMYC2 in tomato, overexpression and RNA interference (RNAi) LeMYC2 tomato transgenic plants were generated. Examination of seedling morphology, physiological responses and light regulated gene expression has revealed that LeMYC2 works as a negative regulator of blue light mediated photomorphogenesis. Furthermore, LeMYC2 specifically binds to the G-box of LeRBCS-3A promoter. Overexpression of LeMYC2 has led to increased root length with more number of lateral roots. The tomato plants overexpressing LeMYC2 have reduced internode distance with more branches, and display the opposite morphology to RNAi transgenic lines. Furthermore, this study shows that LeMYC2 promotes ABA and JA responsiveness.ConclusionsCollectively, this study highlights that working in light, ABA and JA signaling pathways LeMYC2 works as an important regulator for growth and development in tomato plants.

Uncovering DCL1-dependent small RNA loci on plant genomes: a structure-based approach

In plants, Dicer-like 1 (DCL1)-mediated two-step cleavages are essential for the processing of microRNA (miRNA) gene products. Interestingly, DCL1 has been indicated to be involved in the production of many small RNAs (sRNAs) that cannot be classified as canonical miRNAs. However, genomic and functional information on the non-miRNA, DCL1-dependent sRNAs is still limited. Here, we propose a secondary structure-based approach for identification of the precursors containing novel DCL1-dependent sRNA loci. To demonstrate the utility of the workflow: first, 5898 DCL1-dependent sRNAs of 20-24 nucleotides were identified from the sRNA high-throughput sequencing data sets prepared from rice DCL1 RNA interference transgenic lines. Those perfectly mapped to the rice pre-miRNAs (precursor microRNAs) were removed. The remaining 5795 sRNAs were then mapped onto the rice genome, obtaining 30 902 perfectly matched loci belonging to 2310 sRNAs. A total of 4631 clusters of sRNA loci were defined for secondary structure prediction by using RNAfold. The prediction results generated by two algorithms, namely MFE (minimum free energy) and centroid, were manually compared to identify the conserved long-stem structures containing DCL1-dependent sRNA loci. For the purpose of a case study, a portion of the prediction results was screened manually. As a result, 60 clusters displayed great potential for forming featured long-stem structures for the generation of DCL1-dependent sRNAs. Together, the results indicate that the proposed workflow is applicable for the identification of novel DCL1-dependent sRNA loci on plant genomes.

Light-Regulated Hypocotyl Elongation Involves Proteasome-Dependent Degradation of the Microtubule Regulatory Protein WDL3 in Arabidopsis

Light significantly inhibits hypocotyl cell elongation, and dark-grown seedlings exhibit elongated, etiolated hypocotyls. Microtubule regulatory proteins function as positive or negative regulators that mediate hypocotyl cell elongation by altering microtubule organization. However, it remains unclear how plants coordinate these regulators to promote hypocotyl growth in darkness and inhibit growth in the light. Here, we demonstrate that WAVE-DAMPENED 2-LIKE3 (WDL3), a microtubule regulatory protein of the WVD2/WDL family from Arabidopsis thaliana, functions in hypocotyl cell elongation and is regulated by a ubiquitin-26S proteasome-dependent pathway in response to light. WDL3 RNA interference Arabidopsis seedlings grown in the light had much longer hypocotyls than controls. Moreover, WDL3 overexpression resulted in overall shortening of hypocotyl cells and stabilization of cortical microtubules in the light. Cortical microtubule reorganization occurred slowly in cells from WDL3 RNA interference transgenic lines but was accelerated in cells from WDL3-overexpressing seedlings subjected to light treatment. More importantly, WDL3 protein was abundant in the light but was degraded through the 26S proteasome pathway in the dark. Overexpression of WDL3 inhibited etiolated hypocotyl growth in regulatory particle non-ATPase subunit-1a mutant (rpn1a-4) plants but not in wild-type seedlings. Therefore, a ubiquitin-26S proteasome-dependent mechanism regulates the levels of WDL3 in response to light to modulate hypocotyl cell elongation.

Arabidopsis MICROTUBULE DESTABILIZING PROTEIN40 Is Involved in Brassinosteroid Regulation of Hypocotyl Elongation

The brassinosteroid (BR) phytohormones play crucial roles in regulating plant cell growth and morphogenesis, particularly in hypocotyl cell elongation. The microtubule cytoskeleton is also known to participate in the regulation of hypocotyl elongation. However, it is unclear if BR regulation of hypocotyl elongation involves the microtubule cytoskeleton. In this study, we demonstrate that BRs mediate hypocotyl cell elongation by influencing the orientation and stability of cortical microtubules. Further analysis identified the previously undiscovered Arabidopsis thaliana microtubule destabilizing protein40 (MDP40) as a positive regulator of hypocotyl cell elongation. Brassinazole-resistant1, a key transcription factor in the BR signaling pathway, directly targets and upregulates MDP40. Overexpression of MDP40 partially rescued the shorter hypocotyl phenotype in BR-deficient mutant de-etiolated-2 seedlings. Reorientation of the cortical microtubules in the cells of MDP40 RNA interference transgenic lines was less sensitive to BR. These findings demonstrate that MDP40 is a key regulator in BR regulation of cortical microtubule reorientation and mediates hypocotyl growth. This study reveals a mechanism involving BR regulation of microtubules through MDP40 to mediate hypocotyl cell elongation.

Transparent Testa16Plays Multiple Roles in Plant Development and Is Involved in Lipid Synthesis and Embryo Development in Canola

Transparent Testa16 (TT16), a transcript regulator belonging to the B(sister) MADS box proteins, regulates proper endothelial differentiation and proanthocyanidin accumulation in the seed coat. Our understanding of its other physiological roles, however, is limited. In this study, the physiological and developmental roles of TT16 in an important oil crop, canola (Brassica napus), were dissected by a loss-of-function approach. RNA interference (RNAi)-mediated down-regulation of tt16 in canola caused dwarf phenotypes with a decrease in the number of inflorescences, flowers, siliques, and seeds. Fluorescence microscopy revealed that tt16 deficiency affects pollen tube guidance, resulting in reduced fertility and negatively impacting embryo and seed development. Moreover, Bntt16 RNAi plants had reduced oil content and altered fatty acid composition. Transmission electron microscopy showed that the seeds of the RNAi plants had fewer oil bodies than the nontransgenic plants. In addition, tt16 RNAi transgenic lines were more sensitive to auxin. Further analysis by microarray showed that tt16 down-regulation alters the expression of genes involved in gynoecium and embryo development, lipid metabolism, auxin transport, and signal transduction. The broad regulatory function of TT16 at the transcriptional level may explain the altered phenotypes observed in the transgenic lines. Overall, the results uncovered important biological roles of TT16 in plant development, especially in fatty acid synthesis and embryo development.