Mapping and Detection of Genes Related to Trichome Development in Black Gram (Vigna mungo (L.) Hepper).

Black gram (Vigna mungo (L.) Hepper) is a pulses crop with good digestible protein and a high carbohydrate content, so it is widely consumed as human food and animal feed. Trichomes are large, specialized epidermal cells that confer advantages on plants under biotic and abiotic stresses. Genes regulating the development of trichomes are well characterized in Arabidopsis and tomato. However, little is known about trichome development in black gram. In this study, a high-density map with 5734 bin markers using an F2 population derived from a trichome-bearing and a glabrous cultivar of black gram was constructed, and a major quantitative trait locus (QTL) related to trichomes was identified. Six candidate genes were located in the mapped interval region. Fourteen single-nucleotide polymorphisms (SNPs) or insertion/deletions (indels) were associated with those genes. One indel was located in the coding region of the gene designated as Scaffold_9372_HRSCAF_11447.164. Real-time quantitative PCR (qPCR) analysis demonstrated that only one candidate gene, Scaffold_9372_HRSCAF_11447.166, was differentially expressed in the stem between the two parental lines. These two candidate genes encoded the RNA polymerase-associated protein Rtf1 and Bromodomain adjacent to zinc finger domain protein 1A (BAZ1A). These results provide insights into the regulation of trichome development in black gram. The candidate genes may be useful for creating transgenic plants with improved stress resistance and for developing molecular markers for trichome selection in black gram breeding programs.

Isobaric Tags for Relative and Absolute Quantitation-Based Proteomics Analysis Revealed Proteins Involved in Drought Response during the Germination Stage in Faba Bean.

The faba bean, a significant cool-season edible legume crop, is susceptible to drought during the germination stage. Research regarding the genetic regulation of drought tolerance throughout this stage in the faba bean is limited. The differentially expressed proteins (DEPs) in faba beans between the drought-tolerant variety C105 and the drought-sensitive variant E1 during seed germination were identified in this work, accomplished through isobaric tags for relative and absolute quantitation (iTRAQ) analysis. A total of 3827 proteins were identified in the two varieties of germinating seeds. Compared to those of variety E1, an increase in 108 DEPs and a decrease in 61 DEPs were observed in variety C105 under drought. Conversely, in the control group, variety C105 showed 108 significantly upregulated DEPs and 55 significantly downregulated DEPs. GO and KEGG analyses showed that the DEPs associated with glutathione metabolism and protein processing demonstrated significant increases in response to drought stress. Protein-protein interaction (PPI) analysis unveiled three closely connected functional modules of protein translation, DNA replication, and post-translational modification, originating from 22 DEPs derived from the germination period of two varieties under drought stress. To verify the proteomic function, we selected three differentially expressed protein coding genes, which were overexpressed or silenced in tobacco, thereby enhancing the drought resistance of tobacco. This was accompanied via altered levels of superoxide dismutase or peroxidase in transgenic plants under drought stress. The possible mechanism for drought tolerance in germinating seeds of faba bean involves increasing protein translation, decreasing DNA replication, and modifying chromatin. These findings offer invaluable insights into the reaction mechanism in response to drought stress in faba beans. The identified DEPs could be utilized in faba bean breeding initiatives to manage drought.

Thaumatin-like protein family genes VfTLP4-3 and VfTLP5 are critical for faba bean's response to drought stress at the seedling stage.

Thaumatin-like proteins (TLPs) are a diverse family of pathogenesis-related proteins (PR-5) found in various plant species. Faba bean is an economically important crop known for its nutritional value and resilience to harsh environmental conditions, including drought. In this study, we conducted a comprehensive analysis of the gene structure, phylogenetics, and expression patterns of TLP genes in faba bean, with a specific focus on their response to drought stress. A total of 10 TLP genes were identified and characterized from the faba bean transcriptome, which could be classified into four distinct groups based on their evolutionary relationships. Conserved cysteine residues and REDDD motifs, which are characteristic features of TLPs, were found in most of the identified VfTLP members, and these proteins were likely to reside in the cytoplasm. Two genes, VfTLP4-3 and VfTLP5, exhibited significant upregulation under drought conditions. Additionally, ectopically expressing VfTLP4-3 and VfTLP5 in tobacco leaves resulted in enhanced drought tolerance and increased peroxidase (POD) activity. Moreover, the protein VfTLP4-3 was hypothesized to interact with glycoside hydrolase family 18 (GH18), endochitinase, dehydrin, Barwin, and aldolase, all of which are implicated in chitin metabolism. Conversely, VfTLP5 was anticipated to associate with peptidyl-prolyl cis-trans isomerase-like 3, a molecule linked to the synthesis of proline. These findings suggest that these genes may play crucial roles in mediating the drought response in faba bean through the regulation of these metabolic pathways, and serve as a foundation for future genetic improvement strategies targeting enhanced drought resilience in this economically important crop.

Genome-Wide Identification of bZIP Transcription Factors in Faba Bean Based on Transcriptome Analysis and Investigation of Their Function in Drought Response.

Faba bean is an important cool-season edible legume crop that is constantly threatened by abiotic stresses such as drought. The basic leucine zipper (bZIP) gene family is one of the most abundant and diverse families of transcription factors in plants. It regulates plant growth and development and plays an important role in the response to biotic and abiotic stresses. In this study, we identified 18 members of the faba bean bZIP transcription factor family at the genome-wide level based on previous faba bean drought stress transcriptome sequencing data. A phylogenetic tree was constructed to group the 18 VfbZIP proteins into eight clades. Analysis of cis-acting elements in the promoter region suggested that these 18 VfbZIPs may be involved in regulating abiotic stress responses such as drought. Transcriptome data showed high expression of seven genes (VfbZIP1, VfbZIP2, VfbZIP5, VfbZIP7, VfbZIP15, VfbZIP17, and VfbZIP18) in the drought-tolerant cultivar under drought stress, in which VfbZIP1, VfbZIP2, and VfbZIP5 were consistently expressed as detected by quantitative real-time polymerase chain reaction (qRT-PCR) compared to the transcriptome data. Ectopic overexpression of the three VfbZIPs in tobacco, based on the potato Virus X (PVX) vector, revealed that VfbZIP5 enhanced the drought tolerance. Overexpressed VfbZIP5 in plants showed lower levels of proline (PRO), malondialdehyde (MDA), and peroxidase (POD) compared to those overexpressing an empty vector under 10 days of drought stress. Protein-protein interaction (PPI) analysis showed that VfbZIP5 interacted with seven proteins in faba bean, including VfbZIP7 and VfbZIP10. The results depict the importance of VfbZIPs in response to drought stress, and they would be useful for the improvement of drought tolerance.

Genomic analyses of rice bean landraces reveal adaptation and yield related loci to accelerate breeding.

Rice bean (Vigna umbellata) is an underexploited domesticated legume crop consumed for dietary protein in Asia, yet little is known about the genetic diversity of this species. Here, we present a high-quality reference genome for a rice bean landrace (FF25) built using PacBio long-read data and a Hi-C chromatin interaction map, and assess the phylogenetic position and speciation time of rice bean within the Vigna genus. We sequence 440 landraces (two core collections), and GWAS based on data for growth sites at three widely divergent latitudes reveal loci associated with flowering and yield. Loci harboring orthologs of FUL (FRUITFULL), FT (FLOWERING LOCUS T), and PRR3 (PSEUDO-RESPONSE REGULATOR 3) contribute to the adaptation of rice bean from its low latitude center of origin towards higher latitudes, and the landraces which pyramid early-flowering alleles for these loci display maximally short flowering times. We also demonstrate that copy-number-variation for VumCYP78A6 can regulate seed-yield traits. Intriguingly, 32 landraces collected from a mountainous region in South-Central China harbor a recently acquired InDel in TFL1 (TERMINAL FLOWER1) affecting stem determinacy; these materials also have exceptionally high values for multiple human-desired traits and could therefore substantially advance breeding efforts to improve rice bean.

iTRAQ Proteomic Analysis of Wheat (Triticum aestivum L.) Genotypes Differing in Waterlogging Tolerance.

Transient and chronic waterlogging constrains crop production in many regions of the world. Here, we invoke a novel iTRAQ-based proteomic strategy to elicit protein synthesis and regulation responses to waterlogging in tolerant (XM 55) and sensitive genotypes (YM 158). Of the 7,710 proteins identified, 16 were distinct between the two genotypes under waterlogging, partially defining a proteomic basis for waterlogging tolerance (and sensitivity). We found that 11 proteins were up-regulated and 5 proteins were down-regulated; the former included an Fe-S cluster assembly factor, heat shock cognate 70, GTP-binding protein SAR1A-like and CBS domain-containing protein. Down-regulated proteins contained photosystem II reaction center protein H, carotenoid 9, 10 (9', 10')-cleavage dioxygenase-like, psbP-like protein 1 and mitochondrial ATPase inhibitor. We showed that nine proteins responded to waterlogging with non-cultivar specificity: these included 3-isopropylmalate dehydratase large subunit, solanesyl-diphosphate synthase 2, DEAD-box ATP-dependent RNA helicase 3, and 3 predicted or uncharacterized proteins. Sixteen of the 28 selected proteins showed consistent expression patterns between mRNA and protein levels. We conclude that waterlogging stress may redirect protein synthesis, reduce chlorophyll synthesis and enzyme abundance involved in photorespiration, thus influencing synthesis of other metabolic enzymes. Collectively, these factors accelerate the accumulation of harmful metabolites in leaves in waterlogging-susceptible genotypes. The differentially expressed proteins enumerated here could be used as biological markers for enhancing waterlogging tolerance as part of future crop breeding programs.

Effects of three graphene-based materials on the growth and photosynthesis of Brassica napus L.

The environmental safety and threats of graphene-based materials (GBMs) to the agroecosystem have attracted increasing attention in recent years. However, the mechanisms underlying the effects of GBMs on plants remain unclear. Here, we investigated the phytotoxicity of reduced graphene oxide (RGO), graphene oxide (GO) and amine-functionalized graphene (G-NH2) on Brassica napus L. The results revealed that RGO impaired photosynthesis mainly by decreasing the chlorophyll content and Rubisco activity. A further gene-level analysis suggested that this effect of RGO might be due to its toxicity on sulfate transmembrane transporter and nitrogen metabolism, which ultimately led to nutrient imbalance. However, GO directly damaged the photosystem by disrupting the chloroplast structure, and a decrease in Rubisco activity indicated that GO also inhibits carbon fixation. Further gene-level analysis demonstrated that GO has toxicity on the chloroplast membrane, photosystem, photosynthethic electron transport and F-type ATPase. In addition, G-NH2 at 10-1000 mg L-1 showed no significant toxicity. These findings shed light on the potential mechanism for the toxicity of GBMs on plants for risk assessment.

iTRAQ based protein profile analysis revealed key proteins involved in regulation of drought-tolerance during seed germination in Adzuki bean.

Adzuki bean is an important legume crop due to its high-quality protein, fiber, vitamins, minerals as well as rich bioactive substances. However, it is vulnerable to drought at the germination stage. However, little information is available about the genetic control of drought tolerance during seed germination in adzuki bean. In this study, some differential expression proteins (DEPs) were identified during seed germination between the drought-tolerant variety 17235 and drought-sensitive variety 17033 in adzuki bean using iTRAQ method. A total of 2834 proteins were identified in the germinating seeds of these two adzuki beans. Compared with the variety 17033, 87 and 80 DEPs were increased and decreased accumulation in variety 17235 under drought, respectively. Meanwhile, in the control group, a few DEPs, including 9 up-regulated and 21 down-regulated proteins, were detected in variety 17235, respectively. GO, KEGG, and PPI analysis revealed that the DEPs related to carbohydrate metabolism and energy production were significantly increased in response to drought stresses. To validate the proteomic function, the ectopic overexpression of V-ATPase in tobacco was performed and the result showed that V-ATPase upregulation could enhance the drought tolerance of tobacco. The results provide valuable insights into genetic response to drought stress in adzuki bean, and the DEPs could be applied to develop biomarkers related to drought tolerant in adzuki bean breeding projects.

Novel Insights Into Genetic Responses for Waterlogging Stress in Two Local Wheat Cultivars in Yangtze River Basin.

Wheat (Triticum aestivum L.), the most widely cultivated crop, is affected by waterlogging that limited the wheat production. Given the incompleteness of its genome annotation, PacBio SMRT plus Illumina short-read sequencing strategy provided an efficient approach to investigate the genetic regulation of waterlogging stress in wheat. A total of 947,505 full-length non-chimetric (FLNC) sequences were obtained with two wheat cultivars (XM55 and YM158) with PacBio sequencing. Of these, 5,309 long-non-coding RNAs, 1,574 fusion genes and 739 transcription factors were identified with the FLNC sequences. These full-length transcripts were derived from 49,368 genes, including 47.28% of the genes annotated in IWGSC RefSeq v1.0 and 40.86% genes encoded two or more isoforms, while 27.31% genes in the genome annotation of IWGSC RefSeq v1.0 were multiple-exon genes encoding two or more isoforms. Meanwhile, the individuals with waterlogging treatments (WL) and control group (CK) were selected for Illumina sequencing. Totally, 6,829 differentially expressed genes (DEGs) were detected from four pairwise comparisons. Notably, 942 DEGs were overlapped in the two comparisons (i.e., XM55-WL vs. YM158-WL and YM158-WL vs. YM158-CK). Undoubtedly, the genes involved in photosynthesis were downregulated after waterlogging treatment in two cultivars. Notably, the genes related to steroid biosynthesis, steroid hormone biosynthesis, and downstream plant hormone signal transduction were significantly upregulated after the waterlogging treatment, and the YM158 variety revealed different genetic regulation patterns compared with XM55. The findings provided valuable insights into unveiling regulation mechanisms of waterlogging stress in wheat at anthesis and contributed to molecular selective breeding of new wheat cultivars in future.

Characterization of Drought-Responsive Transcriptome During Seed Germination in Adzuki Bean (Vigna angularis L.) by PacBio SMRT and Illumina Sequencing.

The full-length single-molecular sequencing and short reads Illumina sequencing were combined to generate the transcripts of adzuki bean with high-quality. A total of 17,636 loci and 60,454 transcripts were detected in this study. To characterize the drought-responsive genes during seed germination in adzuki bean, two varieties, i.e., tolerant and sensitive to drought stress, were selected to conduct analysis of alternative splicing dynamics (AS) and differentially expressed genes (DEGs) by combining the newly assembled draft genome and public adzuki bean reference genome. AS analysis indicated that both the two varieties underwent a little more AS events under control conditions than under drought stress. Among the AS events, IR (intron retention) predominately accounted for 34.3%, whereas AD (alternative donor site) was the least frequent with 15.8%. Meanwhile, 562 long non-coding RNAs, 409 fusion genes and 1208 transcription factors were identified. Moreover, a total of 5,337 DEGs were identified in comparison of the two varieties with drought or control treatments. Notably, 82 DEGs were discovered in the two varieties under drought stress, which might be the candidate in regulation of seed germination to answer for different drought tolerance. The DEGs encoded proteins involved in primary or second metabolism, plant hormone signal transduction, transcript or translation processes, ubiquitin proteasome system, transcription factor, transporters, and so on. The results facilitate to increase the knowledge about the mechanism of drought tolerance during crop seed germination, and provide reference for the breeding of drought-tolerant adzuki bean.

Transcriptome profile analysis of two Vicia faba cultivars with contrasting salinity tolerance during seed germination.

Faba bean (Vicia faba L.) is an important food legume crop. Salinity soils severely constrain the production of faba bean, however, the seed germination of faba bean, which is a vital plant growth stage, is sensitive to salinity. Planting improved varieties of faba bean, which exhibit salt tolerance in seed germination stage, is an optimal strategy for faba bean product. To investigate the genes dynamics during the seed germination stage under salinity, RNA-seq method was used to investigate genome-wide transcription profiles of two faba bean varieties with contrast salt-tolerance during the seed germination. A total of 4,486 differentially expressed genes (DEGs) were identified among the comparison of salt-tolerant variety Y134 and salt-sensitive variety Y078 treated with salinity or not. Of these, 1,410 candidate DEGs were identified as salt-stress response genes. Furthermore, 623 DEGs were identified as variety-specific response gene during seed germination at 16 h or 24 h with salt treatment. Based on the pathway enrichment according to the Kyoto Encyclopedia of Genes and Genomes database (KEGG), these DEGs involving in cell wall loosening (e.g., xyloglucan endotransglucosylase/hydrolase, chitinase, and expansin), hormone metabolism (e.g., LEA genes, genes associated with ABA or ethylene signal pathway), chromatin remodeling (e.g., chromatin structure proteins, LHP1), small interfering RNA pathway, etc., were significantly up-regulated in salt-tolerance variety with salt treatment, indicating that they play critical roles in regulation of seed germination. The results indicated that a clearer mechanism of gene regulation that regulates the seed germination responding to salinity in faba bean. These findings are helpful to increase the understanding of the salt tolerance mechanism of crops during seed germination, and provide valuable genetic resource for the breeding of salt-tolerant faba bean varieties in future.

Identification of QTL TGW12 responsible for grain weight in rice based on recombinant inbred line population crossed by wild rice (Oryza minuta) introgression line K1561 and indica rice G1025.

BACKGROUND: Limited genetic resource in the cultivated rice may hinder further yield improvement. Some valuable genes that contribute to rice yield may be lost or lacked in the cultivated rice. Identification of the quantitative trait locus (QTL) for yield-related traits such as thousand-grain weight (TGW) from wild rice speices is desired for rice yield improvement. RESULTS: In this study, sixteen TGW QTL were identified from a recombinant inbred line (RIL) population derived from the cross between the introgression line K1561 of Oryza minuta and the rice cultivar G1025. TGW12, One of most effective QTL was mapped to the region of 204.12 kb between the marker 2,768,345 and marker 2,853,491 of the specific locus amplified fragment (SLAF). The origin of TGW12 was tested using three markers nearby or within the TGW12 region, but not clarified yet. Our data indicated thirty-two open reading fragments (ORFs) were present in the region. RT-PCR analysis and sequence alignment showed that the coding domain sequences of ORF12, one MADS-box gene, in G1025 and K1561 were different due to alternative slicing, which caused premature transcription termination. The MADS-box gene was considered as a candidate of TGW12. CONCLUSION: The effective QTL, TGW12, was mapped to a segment of 204.12 kb using RILs population and a MADS-box gene was identified among several candidate genes in the segment. The region of TGW12 should be further narrowed and creation of transgenic lines will reveal the gene function. TGW12 could be applied for improvement of TGW in breeding program.

Identification of a Novel QTL for Panicle Length From Wild Rice (Oryza minuta) by Specific Locus Amplified Fragment Sequencing and High Density Genetic Mapping.

Wild rice possesses a large number of valuable genes that have been lost or do not exist in cultivated rice. To exploit the desirable gene controlling panicle length (PL) in wild rice Oryza minuta, a recombinant inbred line (RIL) population was constructed that was derived from a cross between the long panicle introgression line K1561 with Oryza minuta segments and a short panicle accession G1025. Specific Locus Amplified Fragment (SLAF) sequencing technology was used to uncover single nucleotide polymorphisms (SNPs) and construct the high-density genetic linkage map. Using 201 RIL populations, a high-density genetic map was developed, and spanned 2781.76 cM with an average genetic distance 0.45 cM. The genetic map was composed of 5, 521 markers on 12 chromosomes. Based on this high-density genome map, quantitative trait loci (QTL) for PL were analyzed for 2 years under four environments. Seven QTLs were detected, which were distributed within chromosomes 4, 9, and 10, respectively. pl4.1 was detected twice, and pl10.1 was only detected once. Although pl9.1 was only detected once, it was very near pl9.2 in the genetic map which was detected three times. Thus, we speculate one major QTL exists in the region of pl9.1 and pl9.2 to control PL (temporarily referred to as pl9). pl9 is a potentially novel allele derived from Oryza minuta, and it can be used for genetic improvement of cultivar rice.

Identification of phosphorus deficiency responsive proteins in a high phosphorus acquisition soybean (Glycine max) cultivar through proteomic analysis.

As one of the major oil crops, soybean might be seriously affected by phosphorus deficiency on both yield and quality. Understanding the molecular basis of phosphorus uptake and utilization in soybean may help to develop phosphorus (P) efficient cultivars. On this purpose, we conducted a comparative proteomic analysis on a high P acquisition soybean cultivar BX10 under low and high P conditions. A total of 61 unique proteins were identified as putative P deficiency responsive proteins. These proteins were involved in carbohydrate metabolism, protein biosynthesis/processing, energy metabolism, cellular processes, environmental defense/interaction, nucleotide metabolism, signal transduction, secondary metabolism and other metabolism related processes. Several proteins involved in energy metabolism, cellular processes, and protein biosynthesis and processing were found to be up-regulated in both shoots and roots, whereas, proteins involved in carbohydrate metabolism appeared to be down-regulated. These proteins are potential candidates for improving P acquisition. These findings provide a useful starting point for further research that will provide a more comprehensive understanding of molecular mechanisms through which soybeans adapt to P deficiency condition.

Genetic analysis and molecular mapping of resistance gene to Phakopsora pachyrhizi in soybean germplasm SX6907.

KEY MESSAGE: In this study, Rpp6907, a novel resistance gene/allele to Phakopsora pachyrhizi in soybean, was mapped in a 111.9-kb region, including three NBS-LRR type predicted genes, on chromosome 18. Soybean rust caused by Phakopsora pachyrhizi Sydow has been reported in numerous soybean-growing regions worldwide. The development of rust-resistant varieties is the most economical and environmentally safe method to control the disease. The Chinese soybean germplasm SX6907 is resistant to P. pachyrhizi and exhibits immune reaction compared with the known Rpp genes. These characteristics suggest that SX6907 may carry at least one novel Rpp gene/allele. Three F2 populations from the crosses of SX6907 (resistant) and Tianlong 1, Zhongdou40, and Pudou11 (susceptible) were used to map the Rpp gene. Three resistance responses (immune, red-brown, and tan-colored lesion) were observed from the F2 individuals. The segregation follows a ratio of 1(resistance):2(heterozygous):1(susceptible), indicating that the resistance in SX6907 is controlled by a single incomplete dominant gene (designated as Rpp6907). Results showed that Rpp6907 was mapped on soybean chromosome 18 (molecular linkage group G, MLG G) flanked by simple sequence repeat (SSR) markers SSR24 and SSR40 at a distance of 111.9 kb. Among the ten genes marked within this 111.9-kb region between the two markers, three genes (Glyma18g51930, Glyma18g51950, and Glyma18g51960) are nucleotide-binding site and leucine-rich repeat-type genes. These genes may be involved in recognizing the presence of pathogens and ultimately conferring resistance. Based on resistance spectrum analysis and mapping results, we inferred that Rpp6907 is a novel gene different from Rpp1 in PI 200492, PI 561356, PI 587880A, PI 587886, and PI 594538A, or a new Rpp1-b allele.

Virus-based microRNA silencing in plants.

MicroRNAs (miRNAs) play pivotal roles in various biological processes across kingdoms. Many plant miRNAs have been experimentally identified or predicted by bioinformatics mining of small RNA databases. However, the functions of these miRNAs remain largely unknown due to the lack of effective genetic tools. Here, we report a virus-based microRNA silencing (VbMS) system that can be used for functional analysis of plant miRNAs. VbMS is performed through tobacco rattle virus-based expression of miRNA target mimics to silence endogenous miRNAs. VbMS of either miR172 or miR165/166 caused developmental defects in Nicotiana benthamiana. VbMS of miR319 reduced the complexity of tomato (Solanum lycopersicum) compound leaves. These results demonstrate that tobacco rattle virus-based VbMS is a powerful tool to silence endogenous miRNAs and to dissect their functions in different plant species.

Knockdown of OsHox33, a member of the class III homeodomain-leucine zipper gene family, accelerates leaf senescence in rice.

The class III homeodomain-leucine zipper (HD-Zip III) gene family plays important roles in plant growth and development, including regulation of apical embryo patterning, embryonic shoot meristem formation, leaf polarity, vascular development, and meristem function, with a particularly crucial function in leaf development. Although HD-Zip III members are highly conserved in land plants, previous studies, such as genetic analyses based on multiple mutants in Arabidopsis and other plants, suggest that various HD-Zip III family genes have evolved with distinct functions and pleiotropic effects on plant growth and development. In this study, we analyzed a HD-Zip III member, OsHox33, and demonstrated that it plays an important role in age-dependent leaf senescence in rice. We constructed two specific RNAi vectors derived from the 5'-end region and 3'-UTR of OsHox33 to knockdown its expression. Transgenic plants harboring either RNAi construct displayed similar phenotypes of precocious leaf senescence symptoms, suggesting that knockdown of OsHox33 accelerates leaf senescence in rice. pOsHox33::GUS fusion expression and RT-PCR revealed that OsHox33 is highly expressed in young organs, especially in young meristems such as shoot apical meristems, intercalary meristems, and young callus. In addition, real-time PCR indicated that OsHox33 was more highly expressed in young leaves than in old leaves. To further investigate OsHox33 function, we analyzed chloroplast ultrastructure in different-aged leaves of RNAi plants, and found that OsHox33 knockdown accelerated chloroplast degradation, which is consistent with RNAi phenotypes. Finally, real-time PCR studies showed that OsHox33 can regulate the expression of GS1 and GS2, two senescence-associated genes. Taken together, the work presented here provides new insights into the function of HD-Zip III members in plants.

Elucidation of miRNAs-mediated responses to low nitrogen stress by deep sequencing of two soybean genotypes.

Nitrogen (N) is a major limiting factor in crop production, and plant adaptive responses to low N are involved in many post-transcriptional regulation. Recent studies indicate that miRNAs play important roles in adaptive responses. However, miRNAs in soybean adaptive responses to N limitation have been not reported. We constructed sixteen libraries to identify low N-responsive miRNAs on a genome-wide scale using samples from 2 different genotypes (low N sensitive and low N tolerant) subjected to various periods of low nitrogen stress. Using high-throughput sequencing technology (Illumina-Solexa), we identified 362 known miRNAs variants belonging to 158 families and 90 new miRNAs belonging to 55 families. Among these known miRNAs variants, almost 50% were not different from annotated miRNAs in miRBase. Analyses of their expression patterns showed 150 known miRNAs variants as well as 2 novel miRNAs with differential expressions. These differentially expressed miRNAs between the two soybean genotypes were compared and classified into three groups based on their expression patterns. Predicted targets of these miRNAs were involved in various metabolic and regulatory pathways such as protein degradation, carbohydrate metabolism, hormone signaling pathway, and cellular transport. These findings suggest that miRNAs play important roles in soybean response to low N and contribute to the understanding of the genetic basis of differences in adaptive responses to N limitation between the two soybean genotypes. Our study provides basis for expounding the complex gene regulatory network of these miRNAs.

Comparative proteomic analyses reveal the changes of metabolic features in soybean (Glycine max) pistils upon pollination.

Siphonogamy is a critical process in plant reproductive growth, during which numerous cell-cell interaction events occur between pistil and pollen. Previous studies in Solanaceae, Papaveraceae, and Brassicaceae focusing on pollen-stigma recognition in self-incompatible systems have provided many important views. In this study, we profiled the proteome in soybean mature pistils before and after pollination. Comparative analyses of two-dimensional gel electrophoresis maps from un-pollinated and pollinated pistils were conducted. The results showed that 22 proteins were increased and 36 proteins decreased after pollination. Functional categorization showed that most of them were metabolism- and redox-related proteins. The enhancement of primary metabolism, biosynthesis of pollen tube guidance compounds, and adjustment of redox homeostasis system might be helpful for a successful pollination. Quantitative reverse transcript-polymerase chain reaction analysis implied that the regulation of gene expression might happen at both transcriptional and posttranscriptional levels during pollination. This study will enhance our understanding of pollen-stigma interaction in plant sexual reproductive growth.

Transcript profile of the response of two soybean genotypes to potassium deficiency.

The macronutrient potassium (K) is essential to plant growth and development. Crop yield potential is often affected by lack of soluble K. The molecular regulation mechanism of physiological and biochemical responses to K starvation in soybean roots and shoots is not fully understood. In the present study, two soybean varieties were subjected to low-K stress conditions: a low-K-tolerant variety (You06-71) and a low-K-sensitive variety (HengChun04-11). Eight libraries were generated for analysis: 2 genotypes ×2 tissues (roots and shoots) ×2 time periods [short term (0.5 to 12 h) and long term (3 to 12 d)]. RNA derived from the roots and shoots of these two varieties across two periods (short term and long term) were sequenced and the transcriptomes were compared using high-throughput tag-sequencing. To this end, a large number of clean tags (tags used for analysis after removal of dirty tags) corresponding to distinct tags (all types of clean tags) were identified in eight libraries (L1, You06-71-root short term; L2, HengChun04-11-root short term; L3, You06-71-shoot short term; L4, HengChun04-11-shoot short term; L5, You06-71-root long term; L6, HengChun04-11-root long term; L7, You06-71-shoot long term; L8, HengChun04-11-shoot long term). All clean tags were mapped to the available soybean (Glycine max) transcript database (http://www.soybase.org). Many genes showed substantial differences in expression across the libraries. In total, 5,440 transcripts involved in 118 KEGG pathways were either up- or down-regulated. Fifteen genes were randomly selected and their expression levels were confirmed using quantitative RT-PCR. Our results provide preliminary information on the molecular mechanism of potassium absorption and transport under low-K stress conditions in different soybean tissues.