Shibo Zhang

Leafy cotyledon genes are essential for induction of somatic embryogenesis of Arabidopsis.

The capacity for somatic embryogenesis was studied in lec1, lec2 and fus3 mutants of Arabidopsis thaliana (L.) Heynh. It was found that contrary to the response of wild-type cultures, which produced somatic embryos via an efficient, direct process (65–94% of responding explants), lec mutants were strongly impaired in their embryogenic response. Cultures of the mutants formed somatic embryos at a low frequency, ranging from 0.0 to 3.9%. Moreover, somatic embryos were formed from callus tissue through an indirect route in the lec mutants. Total repression of embryogenic potential was observed in double (lec1 lec2, lec1 fus3, lec2 fus3) and triple (fus3 lec1 lec2) mutants. Additionally, mutants were found to exhibit efficient shoot regenerability via organogenesis from root explants. These results provide evidence that, besides their key role in controlling many different aspects of Arabidopsis zygotic embryogenesis, LEC/FUS genes are also essential for in vitro somatic embryogenesis induction. Furthermore, temporal and spatial patterns of auxin distribution during somatic embryogenesis induction were analyzed using transgenic Arabidopsis plants expressing GUS driven by the DR5 promoter. Analysis of data indicated auxin accumulation was rapid in all tissues of the explants of both wild type and the lec2-1 mutant, cultured on somatic embryogenesis induction medium containing 2,4-D. This observation suggests that loss of embryogenic potential in the lec2 mutant in vitro is not related to the distribution of exogenously applied auxin and LEC genes likely function downstream in auxin-induced somatic embryogenesis.

Expression and Characterization of a Redox-Sensing Green Fluorescent Protein (Reduction-Oxidation-Sensitive Green Fluorescent Protein) in Arabidopsis

Arabidopsis (Arabidopsis thaliana) was transformed with a redox-sensing green fluorescent protein (reduction-oxidation-sensitive green fluorescent protein [roGFP]), with expression targeted to either the cytoplasm or to the mitochondria. Both the mitochondrial and cytosolic forms are oxidation-reduction sensitive, as indicated by a change in the ratio of 510 nm light (green light) emitted following alternating illumination with 410 and 474 nm light. The 410/474 fluorescence ratio is related to the redox potential (in millivolts) of the organelle, cell, or tissue. Both forms of roGFP can be reduced with dithiothreitol and oxidized with hydrogen peroxide. The average resting redox potentials for roots are −318 mV for the cytoplasm and −362 mV for the mitochondria. The elongation zone of the Arabidopsis root has a more oxidized redox status than either the root cap or meristem. Mitochondria are much better than the cytoplasm, as a whole, at buffering changes in redox. The data show that roGFP is redox sensitive in plant cells and that this sensor makes it possible to monitor, in real time, dynamic changes in redox in vivo.

Evolutionary Expansion, Gene Structure, and Expression of the Rice Wall-Associated Kinase Gene Family

The wall-associated kinase (WAK) gene family, one of the receptor-like kinase (RLK) gene families in plants, plays important roles in cell expansion, pathogen resistance, and heavy-metal stress tolerance in Arabidopsis (Arabidopsis thaliana). Through a reiterative database search and manual reannotation, we identified 125 OsWAK gene family members from rice (Oryza sativa) japonica cv Nipponbare; 37 (approximately 30%) OsWAKs were corrected/reannotated from earlier automated annotations. Of the 125 OsWAKs, 67 are receptor-like kinases, 28 receptor-like cytoplasmic kinases, 13 receptor-like proteins, 12 short genes, and five pseudogenes. The two-intron gene structure of the Arabidopsis WAK/WAK-Likes is generally conserved in OsWAKs; however, extra/missed introns were observed in some OsWAKs either in extracellular regions or in protein kinase domains. In addition to the 38 OsWAKs with full-length cDNA sequences and the 11 with rice expressed sequence tag sequences, gene expression analyses, using tiling-microarray analysis of the 20 OsWAKs on chromosome 10 and reverse transcription-PCR analysis for five OsWAKs, indicate that the majority of identified OsWAKs are likely expressed in rice. Phylogenetic analyses of OsWAKs, Arabidopsis WAK/WAK-Likes, and barley (Hordeum vulgare) HvWAKs show that the OsWAK gene family expanded in the rice genome due to lineage-specific expansion of the family in monocots. Localized gene duplications appear to be the primary genetic event in OsWAK gene family expansion and the 125 OsWAKs, present on all 12 chromosomes, are mostly clustered.

Similarity of expression patterns of knotted1 and ZmLEC1 during somatic and zygotic embryogenesis in maize ( Zea mays L.).

Abstract. Expression of knotted1 (kn1) and ZmLEC1, a maize homologue of the ArabidopsisLEAFY COTYLEDON1 (LEC1) was studied using in situ hybridization during in vitro somatic embryogenesis of maize (Zea mays L.) genotype Hi-II. Expression of kn1 was initially detected in a small group of cells (5–10) in the somatic embryo proper at the globular stage, in a specific region where the shoot meristem is initiating at the scutellar stage, and specifically in the shoot meristem at the coleoptilar stage. Expression of ZmLEC1 was strongly detected in the entire somatic embryo proper at the globular stage, gradually less in the differentiating scutellum at the scutellar and coleoptilar stages. The results of analyses show that the expression pattern of kn1 during in vitro somatic embryogenesis of maize is similar to that of kn1 observed during zygotic embryo development in maize. The expression pattern of ZmLEC1 in maize during in vitro development is similar to that of LEC1 in Arabidopsis during zygotic embryo development. These observations indicate that in vitro somatic embryogenesis likely proceeds through similar developmental pathways as zygotic embryo development, after somatic cells acquire competence to form embryos. In addition, based on the ZmLEC1 expression pattern, we suggest that expression of ZmLEC1 can be used as a reliable molecular marker for detecting early-stage in vitro somatic embryogenesis in maize.

Transcription profile analyses identify genes and pathways central to root cap functions in maize.

Affymetrix GeneChips arrayed with about one-half (~23K) of the rice genes were used to profile gene transcription activity in three tissues comprising the maize root tip; the proximal meristem (PM), the quiescent center (QC), and the root cap (RC). Here we analyze the gene transcription profile of the RC, compared to both the PM and the QC, from three biological replicates. In the RC, a total of 669 genes were identified as being differentially upregulated, and 365 differentially downregulated. Real-time quantitative RT-PCR analysis was used to confirm upregulated genes in the RC. In addition, using the technique of laser microdissection (LMD) we localized upregulated gene expression to the lateral RC cells. Taken as a whole, transcription profile analyses revealed the upregulation in the maize RC of clusters of genes linked to major metabolic processes and pathways, including: (1) transport, both the export of carbohydrates and the uptake of nutrients; (2) sensing and responding to (often stressful) biotic and abiotic environmental stimuli; (3) integrating the responses of at least 3 major growth regulators (auxin, ethylene, jasmonic acid); (4) processing the large amount of carbohydrate transported into the RC. Although the profile data are derived using heterologous rice GeneChips, with about half of the total rice gene set, this study, nevertheless, provides a genomic scale characterization of the entire RC, and serves as a new platform from which to advance studies of the network of pathways operating in the maize RC.

Transformation of recalcitrant maize elite inbreds using in vitro shoot meristematic cultures induced from germinated seedlings

Abstract. Two new methods of transformation for recalcitrant maize elite inbreds (B73 and a Pioneer Hi-Bred inbred) were successfully developed using shoot meristematic cultures (SMCs) derived from germinated seedlings. One of the methods – the sector proliferation method – involved in vitro induction and proliferation of SMCs from transgenic sectors. These transgenic sectors derived from the bombardment of shoot apical meristems in immature embryos. Using this method, transgenic T1 and T2 progeny were obtained from the Pioneer Hi-Bred maize inbred, PHTE4. The other method – the SMC method – involved direct bombardment of SMCs. Using the second method, transgenic T1 and T2 progeny were produced from the publicly held maize inbred B73. Cellular and molecular analyses showed that SMCs were mainly induced from the nodal regions within the elongating in vitro stem tissues. The induced SMCs, characterized by large numbers of cells expressing KN1, have the potential to produce multiple adventitious shoot meristems. The use of induction and maintenance media containing higher levels of Cu2+ or Zn2+, not needed in earlier investigations on sweet corn, was found to be critical for the successful in vitro culture and transformation of some maize inbreds.

Expression of CDC2Zm and KNOTTED1 during in-vitro axillary shoot meristem proliferation and adventitious shoot meristem formation in maize (Zea mays L.) and barley (Hordeum vulgare L.)

Abstract. Expression of CDC2Zm and KNOTTED1 (KN1) in maize (Zea mays L.) and their cross-reacting proteins in barley (Hordeum vulgare L.) was studied using immunolocalization during in-vitro axillary shoot meristem proliferation and adventitious shoot meristem formation. Expression of CDC2Zm, a protein involved in cell division, roughly correlated with in-vitro cell proliferation and in the meristematic domes CDC2Zm expression was triggered during in-vitro proliferation. Analysis of the expression of KN1, a protein necessary for maintenance of the shoot meristem, showed that KN1 or KN1-homologue(s) expression was retained in meristematic cells during in-vitro proliferation of axillary shoot meristems. Multiple adventitious shoot meristems appeared to form directly from the KN1- or KN1 homologue(s)-expressing meristematic cells in the in-vitro proliferating meristematic domes. However, unlike Arabidopsis (Arabidopsis thaliana) and tobacco (Nicotiana tabacum) leaves ectopically expressing KN1 (G. Chuck et al., 1996 Plant Cell 8: 1277–1289; N. Sinha et al., 1993 Genes Dev. 7: 787–797), transgenic maize leaves over-expressing KN1 were unable to initiate adventitious shoot meristems on their surfaces either in planta or in vitro. Therefore, expression of KN1 is not the sole triggering factor responsible for inducing adventitious shoot meristem formation from in-vitro proliferating axillary shoot meristems in maize. Our results show that genes critical to cell division and plant development have utility in defining in-vitro plant morphogenesis at the molecular level and, in combination with transformation technologies, will be powerful tools in identifying the fundamental molecular and-or genetic triggering factor(s) responsible for reprogramming of plant cells during plant morphogenesis in-vitro.

Mapping Ds insertions in barley using a sequence-based approach

A transposon tagging system, based upon maize Ac/Ds elements, was developed in barley (Hordeum vulgare subsp. vulgare). The long-term objective of this project is to identify a set of lines with Ds insertions dispersed throughout the genome as a comprehensive tool for gene discovery and reverse genetics. AcTPase and Ds-bar elements were introduced into immature embryos of Golden Promise by biolistic transformation. Subsequent transposition and segregation of Ds away from AcTPase and the original site of integration resulted in new lines, each containing a stabilized Ds element in a new location. The sequence of the genomic DNA flanking the Ds elements was obtained by inverse PCR and TAIL-PCR. Using a sequence-based mapping strategy, we determined the genome locations of the Ds insertions in 19 independent lines using primarily restriction digest-based assays of PCR-amplified single nucleotide polymorphisms and PCR-based assays of insertions or deletions.The proncipal strategy was to identify and map sequence polymorphisms in the regions corresponding to the flanking DNA using the Oregon Wolfe Barley mapping population. The mapping results obtained by the sequence-based approach were confirmed by RFLP analyses in four of the lines. In addition, cloned DNA sequences corresponding to the flanking DNA were used to assign map locations to Morex-derived genomic BAC library inserts, thus integrating genetic and physical maps of barley. BLAST search results indicate that the majority of the transposed Ds elements are found within predicted or known coding sequences. Transposon tagging in barley using Ac/Ds thus promises to provide a useful tool for studies on the functional genomics of the Triticeae.

A Role for Mitochondria in the Establishment and Maintenance of the Maize Root Quiescent Center

Mitochondria in the oxidizing environment of the maize (Zea mays) root quiescent center (QC) are altered in function, but otherwise structurally normal. Compared to mitochondria in the adjacent, rapidly dividing cells of the proximal root tissues, mitochondria in the QC show marked reductions in the activities of tricarboxylic acid cycle enzymes. Pyruvate dehydrogenase activity was not detected in the QC. Use of several mitochondrial membrane potential (ΔΨm) sensing probes indicated a depolarization of the mitochondrial membrane in the QC, which suggests a reduction in the capacity of QC mitochondria to generate ATP and NADH. We postulate that modifications of mitochondrial function are central to the establishment and maintenance of the QC.

Transgenic Cereals: Hordeum vulgare L. (barley)

The development of barley as a crop dates to the earliest agricultural activities of humans, and it remains one of the major cereals grown for feed and food, and for the production of beer. In this century, an understanding and application of quantitative genetic theory has created a genetically elite crop that is divergent from its ancestors. Further improvements in barley cultivare will depend on continued access to useful allelic variability. Sexual hybridization will continue to play an important role in such improvement, but its utility is limited because potentially useful alleles are either linked to undesirable alleles or unavailable because of sexual incompatibilty. The advent of molecular genetics and nonsexual gene transfer offers exciting opportunities to bypass these limitations and to provide access to more diverse sources of genes. Recent developments have added barley to the list of major crops that are amenable to this type of genetic manipulation either through direct DNA transfer (bombardment) or mediated by Agrobacterium tumefaciens. However, significant problems remain, and include: 1) the lack of reproducible, efficient transformation systems for commercial germplasm; 2) the induction of stable genetic and epigenetic changes during the in vitro process; and 3) transgene and transgene expression instability. In this chapter, we will discuss and describe the first systems used for the genetic transformation of barley, introduce and describe the development of new systems for barley transformation, and comment on past and future uses of barley transformation as a tool for basic science and commercial application.