Disrupted oxylipin biosynthesis mitigates pathogen infections and pest infestations in cotton (Gossypium hirsutum).

Cotton (Gossypium hirsutum) is the world's most important fiber crop, critical to global textile industries and agricultural economies. However, cotton yield and harvest quality are undermined by the challenges introduced from invading pathogens and pests. Plant-synthesized oxylipins, specifically 9-hydroxy fatty acids resulting from 9-lipoxygenase activity (9-LOX), enhance the growth and development of many microbes and pests. We hypothesized that targeted disruption of 9-LOX-encoding genes in cotton could bolster crop resilience against prominent agronomic threats. Fusarium oxysporum f. sp. vasinfectum (FOV), Aphis gossypii (cotton aphid), and Tobacco rattle virus induced the expression of 9-oxylipin biosynthesis genes, suggesting that the 9-LOX gene products were susceptibility factors to these stressors. Transiently disrupting the expression of the 9-LOX-encoding genes by virus-induced gene silencing significantly reduced target transcript accumulation, and this correlated with impaired progression of FOV infections and a significant decrease in the fecundity of cotton aphids. These findings emphasize that the cotton 9-LOX-derived oxylipins are leveraged by multiple pathogens and pests to enhance their virulence in cotton, and reducing the expression of 9-LOX-encoding genes can benefit cotton crop vitality.

Cotton Meristem Transcriptomes: Constructing an RNA-Seq Pipeline to Explore Crop Architecture Regulation.

Plants stem cells, known as meristems, specify all patterns of growth and organ size. Differences in meristem activities contribute to diverse shoot architectures. As many architectural traits, such as branching patterns, flowering time, and fruit size, are yield determinants, meristem regulation is of fundamental importance to crop productivity. Cotton (Gossypium spp.) produces our most prevalent natural fiber that finds its way into products ranging from industrial cellulose, medical supplies, and paper currency, to a broad diversity of textiles, not least of which is our clothing. However, the cotton plant has growth habits that challenge management practices and limit harvest yield and quality. Unraveling and leveraging the genetic networks regulating meristem activities offers the potential to overcome these limitations. We use virus-based technologies in cotton to perturb signals regulating meristem fate and size. In this chapter, we describe our pipeline for altering cotton meristem dynamics and preparing, analyzing, and exploring the transcriptomes from isolated meristems.

Altered expression of SELF-PRUNING disrupts homeostasis and facilitates signal delivery to meristems.

Meristem maintenance, achieved through the highly conserved CLAVATA-WUSCHEL (CLV-WUS) regulatory circuit, is fundamental in balancing stem cell proliferation with cellular differentiation. Disruptions to meristem homeostasis can alter meristem size, leading to enlarged organs. Cotton (Gossypium spp.), the world's most important fiber crop, shows inherent variation in fruit size, presenting opportunities to explore the networks regulating meristem homeostasis and to impact fruit size and crop value. We identified and characterized the cotton orthologs of genes functioning in the CLV-WUS circuit. Using virus-based gene manipulation in cotton, we altered the expression of each gene to perturb meristem regulation and increase fruit size. Targeted alteration of individual components of the CLV-WUS circuit modestly fasciated flowers and fruits. Unexpectedly, controlled expression of meristem regulator SELF-PRUNING (SP) increased the impacts of altered CLV-WUS expression on flower and fruit fasciation. Meristem transcriptomics showed SP and genes of the CLV-WUS circuit are expressed independently from each other, suggesting these gene products are not acting in the same path. Virus-induced silencing of GhSP facilitated the delivery of other signals to the meristem to alter organ specification. SP has a role in cotton meristem homeostasis, and changes in GhSP expression increased access of virus-derived signals to the meristem.

Cotton architecture: examining the roles of SINGLE FLOWER TRUSS and SELF-PRUNING in regulating growth habits of a woody perennial crop.

By specifying patterns of determinate and indeterminate growth, FLOWERING LOCUS T/SINGLE FLOWER TRUSS (SFT) and TERMINAL FLOWER 1/SELF-PRUNING (SP) regulate plant architecture. Though well characterized in Arabidopsis, the impacts of these genes on the architectures of diverse crops cultivated in different environments, and their potential to enhance crop productivity and management, are less well addressed. Cotton (Gossypium spp.) is naturally a short-day photoperiodic perennial that is now grown primarily as a day-neutral, annual row crop. Different environments and cultivation practices favor specific growth habits to optimize yield, and in cotton, especially in regions that rely heavily on mechanized harvest, the trend has been to more determinate varieties. Identifying and functionally characterizing SFT and SP homologs in cotton uncovered new aspects of how ratios of indeterminate and determinate growth are balanced, and unraveling their genetic networks emphasized how broadly these gene products affect cotton growth habits.

SINGLE FLOWER TRUSS and SELF-PRUNING signal developmental and metabolic networks to guide cotton architectures.

Patterns of indeterminate and determinate growth specify plant architecture and influence crop productivity. In cotton (Gossypium hirsutum), SINGLE FLOWER TRUSS (SFT) stimulates the transition to flowering and determinate growth, while its closely related antagonist SELF-PRUNING (SP) maintains meristems in indeterminate states to favor vegetative growth. Overexpressing GhSFT while simultaneously silencing GhSP produces highly determinate cotton with reduced foliage and synchronous fruiting. These findings suggest that GhSFT, GhSP, and genes in these signaling networks hold promise for enhancing 'annualized' growth patterns and improving cotton productivity and management. To identify the molecular programs underlying cotton growth habits, we used comparative co-expression networks, differential gene expression, and phenotypic analyses in cotton varieties expressing altered levels of GhSFT or GhSP. Using multiple cotton and tomato datasets, we identified diverse genetic modules highly correlated with SFT or SP orthologs which shared related Gene Ontologies in different crop species. Notably, altering GhSFT or GhSP levels in cotton affected the expression of genes regulating meristem fate and metabolic pathways. Further phenotypic analyses of gene products involved in photosynthesis, secondary metabolism, and cell wall biosynthesis showed that early changes in GhSFT and GhSP levels profoundly impacted later development in distal tissues. Identifying the molecular underpinnings of GhSFT and GhSP activities emphasizes their broad actions in regulating cotton architecture.

Cotton CENTRORADIALIS/TERMINAL FLOWER 1/SELF-PRUNING genes functionally diverged to differentially impact plant architecture.

Genes of the CENTRORADIALIS/TERMINAL FLOWER 1/SELF-PRUNING (CETS) family influence meristem identity by controlling the balance between indeterminate and determinate growth, thereby profoundly impacting plant architecture. Artificial selection during cotton (Gossypium hirsutum) domestication converted photoperiodic trees to the day-neutral shrubs widely cultivated today. To understand the regulation of cotton architecture and exploit these principles to enhance crop productivity, we characterized the CETS gene family from tetraploid cotton. We demonstrate that genes of the TERMINAL FLOWER 1 (TFL1)-like clade show different roles in regulating growth patterns. Cotton has five TFL1-like genes: SELF-PRUNING (GhSP) is a single gene whereas there are two TFL1-like and BROTHER OF FT (BFT)-like genes, and these duplications are specific to the cotton lineage. All genes of the cotton TFL1-like clade delay flowering when ectopically expressed in transgenic Arabidopsis, with the strongest phenotypes failing to produce functional flowers. GhSP, GhTFL1-L2, and GhBFT-L2 rescue the early flowering Attfl1-14 mutant phenotype, and the encoded polypeptides interact with a cotton FD protein. Heterologous promoter::GUS fusions illustrate differences in the regulation of these genes, suggesting that genes of the GhTFL1-like clade may not act redundantly. Characterizations of the GhCETS family provide strategies for nuanced control of plant growth.

Monopodial and sympodial branching architecture in cotton is differentially regulated by the Gossypium hirsutum SINGLE FLOWER TRUSS and SELF-PRUNING orthologs.

Domestication of upland cotton (Gossypium hirsutum) converted it from a lanky photoperiodic perennial to a day-neutral annual row-crop. Residual perennial traits, however, complicate irrigation and crop management, and more determinate architectures are desired. Cotton simultaneously maintains robust monopodial indeterminate shoots and sympodial determinate shoots. We questioned if and how the FLOWERING LOCUS T/SINGLE FLOWER TRUSS (SFT)-like and TERMINAL FLOWER1/SELF-PRUNING (SP)-like genes control the balance of monopodial and sympodial growth in a woody perennial with complex growth habit. Virus-based manipulation of GhSP and GhSFT expression enabled unprecedented functional analysis of cotton development. GhSP maintains growth in all apices; in its absence, both monopodial and sympodial branch systems terminate precociously. GhSFT encodes a florigenic signal stimulating rapid onset of sympodial branching and flowering in side shoots of wild photoperiodic and modern day-neutral accessions. High florigen concentrations did not alter monopodial apices, implying that once a cotton apex is SP-determined, it cannot be reset by florigen. GhSP is also essential to establish and maintain cambial activity. Dynamic changes in GhSFT and GhSP levels navigate meristems between monopodial and sympodial programs in a single plant. SFT and SP influenced cotton domestication and are ideal targets for further agricultural optimization.

Phloem-mobile signals affecting flowers: applications for crop breeding.

Transport of endogenous macromolecules within and between tissues serves as a signaling pathway to regulate numerous aspects of plant growth. The florigenic FT gene product moves via the phloem from leaves to apical tissues and induces the flowering program in meristems. Similarly, short interfering RNA (siRNA) signals produced in source or sink tissues move cell-to-cell and long distance via the phloem to apical tissues. Recent advances in identifying these mobile signals regulating flowering or the epigenetic status of targeted tissues can be applicable to crop-breeding programs. In this review, we address the identity of florigen, the mechanism of allocation, and how virus-induced flowering and grafting of transgenes producing siRNA signals affecting meiosis can produce transgene-free progenies useful for agriculture.

Overexpression of FT in cotton affects architecture but not floral organogenesis.

Flowering marks the change from indeterminate to determinate plant growth, and this developmental transition involves the activity of the Arabidopsis FLOWERING LOCUS T (FT) gene product and its orthologs. We demonstrated that when FT is ectopically expressed from a viral vector in cotton, a process referred to as virus induced flowering (VIF), it uncouples flowering from photoperiodic regulation and promotes determinate growth in aerial organs. The accelerated switch to determinate growth affected cotton floral buds and sympodial growth, but did not disrupt floral organogenesis. These results can be interpreted in the context of the balance model, which argues that the balance of indeterminate and determinate growth is influenced by the relative abundance of indeterminate and determinate factors in the growing apices. These results emphasize the expanding role of FT in affecting general determinate growth.

Geminivirus-mediated delivery of florigen promotes determinate growth in aerial organs and uncouples flowering from photoperiod in cotton.

BACKGROUND: Plant architecture and the timing and distribution of reproductive structures are fundamental agronomic traits shaped by patterns of determinate and indeterminate growth. Florigen, encoded by FLOWERING LOCUS T (FT) in Arabidopsis and SINGLE FLOWER TRUSS (SFT) in tomato, acts as a general growth hormone, advancing determinate growth. Domestication of upland cotton (Gossypium hirsutum) converted it from a lanky photoperiodic perennial to a highly inbred, compact day-neutral plant that is managed as an annual row-crop. This dramatic change in plant architecture provides a unique opportunity to analyze the transition from perennial to annual growth. METHODOLOGY/PRINCIPAL FINDINGS: To explore these architectural changes, we addressed the role of day-length upon flowering in an ancestral, perennial accession and in a domesticated variety of cotton. Using a disarmed Cotton leaf crumple virus (CLCrV) as a transient expression system, we delivered FT to both cotton accessions. Ectopic expression of FT in ancestral cotton mimicked the effects of day-length, promoting photoperiod-independent flowering, precocious determinate architecture, and lanceolate leaf shape. Domesticated cotton infected with FT demonstrated more synchronized fruiting and enhanced "annualization". Transient expression of FT also facilitated simple crosses between wild photoperiodic and domesticated day-neutral accessions, effectively demonstrating a mechanism to increase genetic diversity among cultivated lines of cotton. Virus was not detected in the F(1) progeny, indicating that crosses made by this approach do not harbor recombinant DNA molecules. CONCLUSIONS: These findings extend our understanding of FT as a general growth hormone that regulates shoot architecture by advancing organ-specific and age-related determinate growth. Judicious manipulation of FT could benefit cotton architecture to improve crop management.

Manipulating plant architecture with members of the CETS gene family.

The shape or architecture of a plant is specified through the activities of indeterminate and determinate meristems, and the sum of these events sharply impacts plant growth habit, productivity, and crop management. The CENTRORADIALIS/TERMINAL FLOWER 1/SELF-PRUNING (CETS) gene family shares homology to phosphatidylethanolamine binding protein (PEBP) genes and is prominent in controlling the timing and location of the developmental transition from indeterminate to determinate growth, with different family members balancing the activities of others through antagonistic functions. The CETS members FLOWERING LOCUS T (FT) of Arabidopsis and related genes (e.g. SINGLE FLOWER TRUSS, SFT, in tomato) are important in promoting the transition to determinate growth while TERMINAL FLOWER 1 (TFL1) and its homologs (e.g. tomato SELF PRUNING, SP) oppose this activity by maintaining meristems in an indeterminate state. FT orthologs, and perhaps other CETS family members, act as mobile proteinaceous hormones, and can amplify their impact by accumulating in recipient organs. A universal model is emerging for the timing and placement of determinate and indeterminate growth through a balance of FT-like and TFL1-like gene activities, and it is now clear that the domestication of many wild exotics into crops with desired growth habits resulted from selection of altered FT/TFL1 balances. Manipulating this ratio further, through transgenic or viral-based technologies, holds promise for improved agricultural sustainability.

Arabidopsis plants harbouring a mutation in AtSUC2, encoding the predominant sucrose/proton symporter necessary for efficient phloem transport, are able to complete their life cycle and produce viable seed.

BACKGROUND AND AIMS: AtSUC2 encodes a sucrose/proton symporter that localizes throughout the collection and transport phloem and is necessary for efficient transport of sucrose from source to sink tissues in Arabidopsis thaliana. Plants harbouring homozygous AtSUC2 null alleles accumulate sugar, starch, and anthocyanin in mature leaves, have severely delayed development and stunted growth and, in previous studies, failed to complete their life cycle by producing viable seed. METHODS: An AtSUC2 allele with a T-DNA insertion in the second intron was analysed. Full-length transcript from this allele is not produced, and a truncated protein translated from sequences upstream of the insertion site did not catalyse sucrose uptake into yeast, supporting the contention that this is a null allele. Mutant plants were grown in a growth chamber with a diurnal light/dark cycle, and growth patterns recorded. KEY RESULTS: This allele (SALK_038124, designated AtSUC2-4) has the hallmarks of previously described null alleles but, despite compromised carbon partitioning and growth, produces viable seeds. The onset of flowering was chronologically delayed but occurred at the same point in the plastochron index as wild type. CONCLUSIONS: AtSUC2 is important for phloem loading and is therefore fundamental to phloem transport and plant productivity, but plants can complete their life cycle and produce viable seed in its absence. Arabidopsis appears to have mechanisms for mobilizing reduced carbon from the phloem into developing seeds independent of AtSUC2.

A DNA element between At4g28630 and At4g28640 confers companion-cell specific expression following the sink-to-source transition in mature minor vein phloem.

The collection phloem in minor veins is distinct from other vein classes in that the minor veins mature during the sink to source transition and are the primary sites of phloem loading. After maturation, minor vein phloem maintains its character in part through minor-vein specific regulatory cascades; however despite its physiological significance, little of these developmental programs is understood. From an Arabidopsis enhancer trap screen, we identified MATURE MINOR VEIN ELEMENT1 (MMVE1) in the intergenic region between two oppositely oriented genes, the ABC transporter ATM1 (At4g28630) and IAA11 (At4g28640). MMVE1 promotes reporter gene activity in minor vein phloem in a pattern resembling the sink to source transition. Promoter truncation experiments and phylogenetic footprinting demonstrate sequences proximal to ATM1 promote minor vein expression whereas sequences closer to IAA11 repress it. Both orientations of the promoter were used to drive expression of CONSTANS to generate a phloem mobile signal conferring early flowering under non-inductive conditions. Tandem copies of MMVE1 increase minor vein expression strength and specificity. MMVE1 is the first minor vein enhancer characterized from a species that loads from the apoplast, and supports the presence of unique regulatory cascades operating in minor vein phloem.

A novel Arabidopsis acetyltransferase interacts with the geminivirus movement protein NSP.

Protein acetylation is important in regulating DNA-templated processes specifically and protein-protein interactions more generally in eukaryotes. The geminivirus movement protein NSP is essential for virus movement, shuttling the viral DNA genome between the nucleus and the cytoplasm. We have identified a novel Arabidopsis protein, AtNSI, that interacts with NSP. AtNSI is highly conserved among widely divergent plants. Biochemical studies show that its interaction with NSP is direct and that AtNSI acetylates histones, but not NSP, in vitro. Rather, AtNSI specifically acetylates the viral coat protein. AtNSI is a nuclear protein but does not act as a transcriptional coactivator in vitro, which distinguishes it from known eukaryotic histone acetyltransferases. Its overexpression enhances the efficiency of infection by Cabbage leaf curl virus. These findings suggest a role for protein acetylation in coordinating replication of the viral DNA genome with its export from the nucleus.