Anjanasree K. Neelakandan

Recent progress in the understanding of tissue culture-induced genome level changes in plants and potential applications

In vitro cell and tissue-based systems have tremendous potential in fundamental research and for commercial applications such as clonal propagation, genetic engineering and production of valuable metabolites. Since the invention of plant cell and tissue culture techniques more than half a century ago, scientists have been trying to understand the morphological, physiological, biochemical and molecular changes associated with tissue culture responses. Establishment of de novo developmental cell fate in vitro is governed by factors such as genetic make-up, stress and plant growth regulators. In vitro culture is believed to destabilize the genetic and epigenetic program of intact plant tissue and can lead to chromosomal and DNA sequence variations, methylation changes, transposon activation, and generation of somaclonal variants. In this review, we discuss the current status of understanding the genomic and epigenomic changes that take place under in vitro conditions. It is hoped that a precise and comprehensive knowledge of the molecular basis of these variations and acquisition of developmental cell fate would help to devise strategies to improve the totipotency and embryogenic capability in recalcitrant species and genotypes, and to address bottlenecks associated with clonal propagation.

Molecular characterization of stress-inducible GmNAC genes in soybean

Drought is detrimental to plant growth and development, and often results in significant losses to the yields of economically important crops such as soybeans (Glycine max L.). NAC transcription factors (TFs), which consist of a large family of plant-specific TFs, have been reported to enhance drought tolerance in a number of plants. In this study, 31 unigenes that contain the complete open reading frames encoding GmNAC proteins were identified and cloned from soybean. Analysis of C-terminal regulatory domain using yeast one-hybrid system indicated that among 31 GmNAC proteins, 28 have transcriptional activation activity. Expression analysis of these GmNAC genes showed that they are differentially expressed in different organs, suggesting that they have diverse functions during plant growth and development. To search for the drought-inducible GmNAC genes, we prescreened and re-confirmed by quantitative real-time PCR analysis that nine GmNAC genes are induced by dehydration stress with differential induction levels in both shoot and root. The expression profiles of these nine GmNAC genes were also examined under other stresses such as high salinity, cold and with abscisic acid hormone treatments. Phylogenetic analysis of the GmNAC proteins with previously reported drought-inducible NAC proteins of Arabidopsis and rice revealed that the nine drought-inducible GmNAC proteins belong to the “stress-inducible” NAC group. The results of this systematic analysis of the GmNAC family will provide novel tools and resources for the development of improved drought tolerant transgenic soybean cultivars.

An Agrobacterium-delivered CRISPR/Cas9 system for high-frequency targeted mutagenesis in maize.

Summary CRISPR/Cas9 is a powerful genome editing tool in many organisms, including a number of monocots and dicots. Although the design and application of CRISPR/Cas9 is simpler compared to other nuclease‐based genome editing tools, optimization requires the consideration of the DNA delivery and tissue regeneration methods for a particular species to achieve accuracy and efficiency. Here, we describe a public sector system, ISU Maize CRISPR, utilizing Agrobacterium‐delivered CRISPR/Cas9 for high‐frequency targeted mutagenesis in maize. This system consists of an Escherichia coli cloning vector and an Agrobacterium binary vector. It can be used to clone up to four guide RNAs for single or multiplex gene targeting. We evaluated this system for its mutagenesis frequency and heritability using four maize genes in two duplicated pairs: Argonaute 18 (ZmAgo18a and ZmAgo18b) and dihydroflavonol 4‐reductase or anthocyaninless genes (a1 and a4). T0 transgenic events carrying mono‐ or diallelic mutations of one locus and various combinations of allelic mutations of two loci occurred at rates over 70% mutants per transgenic events in both Hi‐II and B104 genotypes. Through genetic segregation, null segregants carrying only the desired mutant alleles without the CRISPR transgene could be generated in T1 progeny. Inheritance of an active CRISPR/Cas9 transgene leads to additional target‐specific mutations in subsequent generations. Duplex infection of immature embryos by mixing two individual Agrobacterium strains harbouring different Cas9/gRNA modules can be performed for improved cost efficiency. Together, the findings demonstrate that the ISU Maize CRISPR platform is an effective and robust tool to targeted mutagenesis in maize.

RNA Interference Knockdown of BRASSINOSTEROID INSENSITIVE1 in Maize Reveals Novel Functions for Brassinosteroid Signaling in Controlling Plant Architecture

Brassinosteroid signaling is central to controlling auricle development and establishing the blade-sheath boundary in maize leaf. Brassinosteroids (BRs) are plant hormones involved in various growth and developmental processes. The BR signaling system is well established in Arabidopsis (Arabidopsis thaliana) and rice (Oryza sativa) but poorly understood in maize (Zea mays). BRASSINOSTEROID INSENSITIVE1 (BRI1) is a BR receptor, and database searches and additional genomic sequencing identified five maize homologs including duplicate copies of BRI1 itself. RNA interference (RNAi) using the extracellular coding region of a maize zmbri1 complementary DNA knocked down the expression of all five homologs. Decreased response to exogenously applied brassinolide and altered BR marker gene expression demonstrate that zmbri1-RNAi transgenic lines have compromised BR signaling. zmbri1-RNAi plants showed dwarf stature due to shortened internodes, with upper internodes most strongly affected. Leaves of zmbri1-RNAi plants are dark green, upright, and twisted, with decreased auricle formation. Kinematic analysis showed that decreased cell division and cell elongation both contributed to the shortened leaves. A BRASSINOSTEROID INSENSITIVE1-ETHYL METHANESULFONATE-SUPPRESSOR1-yellow fluorescent protein (BES1-YFP) transgenic line was developed that showed BR-inducible BES1-YFP accumulation in the nucleus, which was decreased in zmbri1-RNAi. Expression of the BES1-YFP reporter was strong in the auricle region of developing leaves, suggesting that localized BR signaling is involved in promoting auricle development, consistent with the zmbri1-RNAi phenotype. The blade-sheath boundary disruption, shorter ligule, and disrupted auricle morphology of RNAi lines resemble KNOTTED1-LIKE HOMEOBOX (KNOX) mutants, consistent with a mechanistic connection between KNOX genes and BR signaling.

Cloning, functional expression and phylogenetic analysis of plant sterol 24C-methyltransferases involved in sitosterol biosynthesis

Sterol 24C-methyltransferases (SMTs) constitute a group of sequence-related proteins that catalyze the distinct patterns of 24-alkyl sterols that occur throughout nature. Two SMT cDNAs (SMT2-1 and SMT2-2) were cloned by homology based PCR methods from young leaves of Glycine max (soybean) and the corresponding enzymes were expressed functionally in Escherichia coli. The full-length cDNA for SMT2-1 and SMT2-2 have open reading frames of 1086 bp and 1092 bp, respectively, and encode proteins of 361 and 363 residues with a calculated molecular mass of 40.3 and 40.4 kDa, respectively. The substrate preference of the two isoforms was similar yet they differed from SMT1; kinetically SMT2-1 and SMT2-2 generated k(cat) values for the optimal substrate 24(28)methylene lophenol of 0.8 min(-1) and 1.34 min(-1), respectively, compared to the activity of SMT1 that generated a k(cat) for the optimal substrate cycloartenol of 0.6 min(-1). SMT2-2 was purified to homogeneity and the subunit organization shown to be tetrameric in similar fashion to other cloned SMTs. Analysis of the accumulated products catalyzed by the recombinant enzymes demonstrated that soybean SMT2-1 and SMT2-2 operate transalkylation activities analogous to the soybean plant SMT1. Metabolite analyses correlated with transcript profiling of the three SMT isoforms during soybean maturation clearly demonstrated that SMT isoform expression determines specific C24-methyl to C24-ethyl ratios to flowering whereas with seed development there is a disconnection such that the SMT transcript levels decrease against an increase in sterol content; generally SMT2-2 is expressed more than SMT2-1 or SMT1. These observations suggest that the genes that encode SMT1 and SMT2 in sitosterol biosynthesis may have undergone divergent evolution. In support of this proposition, the genomic organization for SMT1 of fungi and protozoa align very closely with one another and to those of the plant SMT2; both sets of SMTs lack introns. Unexpectedly, the SMT1 from Glycine max and other embryophytes of diverse origin possess disparate intron-exon characteristics that can be shown relates back to the algae. Our results suggest that the order of SMT1 appearing before SMT2 in phytosterol synthesis arose recently in plant evolution in response to duplication of a more primitive SMT gene likely to have been bifunctional and catalytically promiscuous.

Molecular characterization and functional analysis of Glycine max sterol methyl transferase 2 genes involved in plant membrane sterol biosynthesis

Sterol C24 methyltransferase (SMT2) genes governing the pattern of phytosterols synthesized in higher plants have been studied in Glycine seedlings and wild-type and engineered Arabidopsis thaliana plants. The SMT2 genes of soybean (SMT2-1 and SMT2-2) previously cloned and characterized (Neelakandan et al. 2009) were shown to complement the SMT deficient cvp1 mutant Arabidopsis plants, consistent with their role in regulation of 24-alkyl sterol-controlled plant physiology. Further analysis of these genes showed that environmental cues, including dehydration, cold, and abscisic acid induced differential changes in transcript levels of the SMT2 during soybean seedling growth. Sterol analyses of transgenic Arabidopsis seeds originating in variant constructs of AtHMGR1, GmSMT1, and GmSMT2 engineered in seeds showed relevant modifications in the ratio of 24-methyl to 24-ethyl sterol in the direction of sitosterol formation. To provide insight into the structural features of the sterol gene that affects transcript regulation, the upstream promoter sequences of soybean SMT2 genes were cloned and characterized. Sequence analysis revealed several important cis-elements and transcription factor binding sites. The analysis of promoter-GUS fusions in transgenic Arabidopsis plants revealed shared and distinct expression features in different developmental stages and tissues. The data are interpreted to imply that SMT2 is an important contributor to normal plant growth and development.

Higher Plant Cytochrome b5 Polypeptides Modulate Fatty Acid Desaturation

Background Synthesis of polyunsaturated fatty acids (PUFAs) in the endoplasmic reticulum of plants typically involves the fatty acid desaturases FAD2 and FAD3, which use cytochrome b5 (Cb5) as an electron donor. Higher plants are reported to have multiple isoforms of Cb5, in contrast to a single Cb5 in mammals and yeast. Despite the wealth of information available on the roles of FAD2 and FAD3 in PUFA synthesis, information regarding the contributions of various Cb5 isoforms in desaturase-mediated reactions is limited. Results The present functional characterization of Cb5 polypeptides revealed that all Arabidopsis Cb5 isoforms are not similarly efficient in ω-6 desaturation, as evidenced by significant variation in their product outcomes in yeast-based functional assays. On the other hand, characterization of Cb5 polypeptides of soybean (Glycine max) suggested that similar ω-6 desaturation efficiencies were shared by various isoforms. With regard to ω-3 desaturation, certain Cb5 genes of both Arabidopsis and soybean were shown to facilitate the accumulation of more desaturation products than others when co-expressed with their native FAD3. Additionally, similar trends of differential desaturation product accumulation were also observed with most Cb5 genes of both soybean and Arabidopsis even if co-expressed with non-native FAD3. Conclusions The present study reports the first description of the differential nature of the Cb5 genes of higher plants in fatty acid desaturation and further suggests that ω-3/ω-6 desaturation product outcome is determined by the nature of both the Cb5 isoform and the fatty acid desaturases.

The naked endosperm Genes Encode Duplicate INDETERMINATE Domain Transcription Factors Required for Maize Endosperm Cell Patterning and Differentiation

A duplicated pair of transcription factors control cell patterning and cell differentiation in maize endosperm development. The aleurone is the outermost layer of cereal endosperm and functions to digest storage products accumulated in starchy endosperm cells as well as to confer important dietary health benefits. Whereas normal maize (Zea mays [Zm]) has a single aleurone layer, naked endosperm (nkd) mutants produce multiple outer cell layers of partially differentiated cells that show sporadic expression of aleurone identity markers such as a viviparous1 promoter-β-glucuronidase transgene. The 15:1 F2 segregation ratio suggested that two recessive genes were involved, and map-based cloning identified two homologous genes in duplicated regions of the genome. The nkd1 and nkd2 genes encode the INDETERMINATE1 domain (IDD) containing transcription factors ZmIDDveg9 and ZmIDD9 on chromosomes 2 and 10, respectively. Independent mutant alleles of nkd1 and nkd2, as well as nkd2-RNA interference lines in which both nkd genes were knocked down, also showed the nkd mutant phenotype, confirming the gene identities. In wild-type kernels, the nkd transcripts were most abundant around 11 to 16 d after pollination. The NKD proteins have putative nuclear localization signals, and green fluorescent protein fusion proteins showed nuclear localization. The mutant phenotype and gene identities suggest that NKD controls a gene regulatory network involved in aleurone cell fate specification and cell differentiation.

Metabolic engineering of soybean affords improved phytosterol seed traits

Different combinations of three rate-limiting enzymes in phytosterol biosynthesis, the Arabidopsis thaliana hydroxyl methylglutaryl CoA1 (HMGR1) catalytic subunit linked to either constitutive or seed-specific β-conglycinin promoter, and the Glycine max sterol methyltransferase1 (SMT1) and sterol methyltransferase2-2 (SMT2-2) genes, under the control of seed-specific Glycinin-1 and Beta-phaseolin promoters, respectively, were engineered in soybean plants. Mature seeds of transgenic plants displayed modest increases in total sterol content, which points towards a tight control of phytosterol biosynthesis. However, in contrast to wild-type seeds that accumulated about 35% of the total sterol in the form of intermediates, in the engineered seeds driven by a seed-specific promoter, metabolic flux was directed to Δ(5) -24-alkyl sterol formation (99% of total sterol). The engineered effect of end-product sterol (sitosterol, campesterol, and stigmasterol) over-production in soybean seeds resulted in an approximately 30% increase in overall sitosterol synthesis, a desirable trait for oilseeds and human health. In contradistinction, increased accumulation of cycloartenol and 24(28)-methylencylartanol (55% of the total sterol) was detected in plants harbouring the constitutive t-HMGR1 gene, consistent with the previous studies. Our results support the possibility that metabolic flux of the phytosterol family pathway is differentially regulated in leaves and seeds.

NKD Transcription Factors Are Central Regulators of Maize Endosperm Development

The NKD transcription factors regulate diverse and key pathways in developing maize endosperm, including cell fate, storage compound accumulation, and seed maturation. NAKED ENDOSPERM1 (NKD1) and NKD2 are duplicate INDETERMINATE DOMAIN (IDD) transcription factors important for maize (Zea mays) endosperm development. RNA-seq analysis of the nkd1 nkd2 mutant endosperm revealed that NKD1 and NKD2 influence 6.4% of the transcriptome in developing aleurone and 6.7% in starchy endosperm. Processes regulated by NKD1 and NKD2 include gene expression, epigenetic functions, cell growth and division, hormone pathways, and resource reserve deposition. The NKD1 and NKD2 proteins bind a consensus DNA sequence of TTGTCGT with slightly different properties. This motif was enriched in the promoters of gene transcripts differentially expressed (DE) in mutant endosperm. DE genes with a NKD binding motif in the 5′ promoter region were considered as likely direct targets of NKD1 and NKD2 regulation, and these putative direct target genes were notably enriched for storage proteins. Transcription assays demonstrate that NKD1 and NKD2 can directly regulate gene transcription, including activation of opaque2 and viviparous1 promoters. NKD2 functions as a negative regulator of nkd1 transcription, consistent with previously reported feedback regulation. NKD1 and NKD2 can homo- and heterodimerize through their ID domains. These analyses implicate NKD1 and NKD2 as central regulators of gene expression in developing maize endosperm.