Solanum lycopersicum CLASS-II KNOX genes regulate fruit anatomy via gibberellin-dependent and independent pathways.

The pericarp is the predominant tissue determining the structural characteristics of most fruits. However, the molecular and genetic mechanisms controlling pericarp development remain only partially understood. Previous studies have identified that CLASS-II KNOX genes regulate fruit size, shape, and maturation in Arabidopsis thaliana and Solanum lycopersicum. Here we characterized the roles of the S. lycopersicum CLASS-II KNOX (TKN-II) genes in pericarp development via a detailed histological, anatomical, and karyotypical analysis of TKN-II gene clade mRNA-knockdown (35S:amiR-TKN-II) fruits. We identify that 35S:amiR-TKN-II pericarps contain more cells around their equatorial perimeter and fewer cell layers than the control. In addition, the cell sizes but not the ploidy levels of these pericarps were dramatically reduced. Further, we demonstrate that fruit shape and pericarp layer number phenotypes of the 35S:amiR-TKN-II fruits can be overridden by the procera mutant, known to induce a constitutive response to the plant hormone gibberellin. However, neither the procera mutation nor exogenous gibberellin application can fully rescue the reduced pericarp width and cell size phenotype of 35S:amiR-TKN-II pericarps. Our findings establish that TKN-II genes regulate tomato fruit anatomy, acting via gibberellin to control fruit shape but utilizing a gibberellin-independent pathway to control the size of pericarp cells.

Tuning of meristem maturation rate increases yield in multiple Triticum aestivum cultivars.

Breeding programs aim to improve crop yield and environmental stability for enhanced food security. The principal methodology in breeding for stable yield gain relies on the indirect selection of beneficial genetics by yield evaluation across diverse environmental conditions. This methodology requires substantial resources while delivering a slow pace of yield gain and environmental adaptation. Alternative methods are required to accelerate gain and adaptation, becoming even more imperative in a changing climate. New molecular tools and approaches can enable accelerated creation and deployment of multiple alleles of genes identified to control key traits. With the advent of tools that enable breeding by targeted allelic selection, identifying gene targets associated with an improved crop performance ideotype will become crucial. Previous studies have shown that altered photoperiod regimes increase yield in wheat (Triticum aestivum). In the current study, we have employed such treatments to study the resulting yield ideotype in five spring wheat cultivars. We found that the photoperiod treatment creates a yield ideotype arising from delayed spike establishment rates that are accompanied by increased early shoot expression of TARGET OF EAT1 (TaTOE1) genes. Genes identified in this way could be used for ideotype-based improve crop performance through targeted allele creation and selection in relevant environments.

CLASS-II KNOX genes coordinate spatial and temporal ripening in tomato.

Fruits can be divided into dry and fleshy types. Dry fruits mature through senescence and fleshy fruits through ripening. Previous studies have indicated that partially common molecular networks could govern fruit maturation in these different fruit types. However, the nature of such networks remains obscure. CLASS-II KNOX genes were shown to regulate the senescence of the Arabidopsis (Arabidopsis thaliana) dry fruits, the siliques, but their roles in fleshy-fruit development are unknown. Here, we investigated the roles of the tomato (Solanum lycopersicum) CLASS-II KNOX (TKN-II) genes in fleshy fruit ripening using knockout alleles of individual genes and an artificial microRNA line (35S:amiR-TKN-II) simultaneously targeting all genes. 35S:amiR-TKN-II plants, as well as a subset of tkn-II single and double mutants, have smaller fruits. Strikingly, the 35S:amiR-TKN-II and tknII3 tknII7/+ fruits showed early ripening of the locular domain while their pericarp ripening was stalled. Further examination of the ripening marker-gene RIPENING INHIBITOR (RIN) expression and 35S:amiR-TKN-II rin-1 mutant fruits suggested that TKN-II genes arrest RIN activity at the locular domain and promote it in the pericarp. These findings imply that CLASS-II KNOX genes redundantly coordinate maturation in both dry and fleshy fruits. In tomato, these genes also control spatial patterns of fruit ripening, utilizing differential regulation of RIN activity at different fruit domains.

Targeted suppression of gibberellin biosynthetic genes ZmGA20ox3 and ZmGA20ox5 produces a short stature maize ideotype.

Maize is one of the world's most widely cultivated crops. As future demands for maize will continue to rise, fields will face ever more frequent and extreme weather patterns that directly affect crop productivity. Development of environmentally resilient crops with improved standability in the field, like wheat and rice, was enabled by shifting the architecture of plants to a short stature ideotype. However, such architectural change has not been implemented in maize due to the unique interactions between gibberellin (GA) and floral morphology which limited the use of the same type of mutations as in rice and wheat. Here, we report the development of a short stature maize ideotype in commercial hybrid germplasm, which was generated by targeted suppression of the biosynthetic pathway for GA. To accomplish this, we utilized a dominant, miRNA-based construct expressed in a hemizygous state to selectively reduce expression of the ZmGA20ox3 and ZmGA20ox5 genes that control GA biosynthesis primarily in vegetative tissues. Suppression of both genes resulted in the reduction of GA levels leading to inhibition of cell elongation in internodal tissues, which reduced plant height. Expression of the miRNA did not alter GA levels in reproductive tissues, and thus, the reproductive potential of the plants remained unchanged. As a result, we developed a dominant, short-stature maize ideotype that is conducive for the commercial production of hybrid maize. We expect that the new maize ideotype would enable more efficient and more sustainable maize farming for a growing world population.

I am all ears: Maximize maize doubled haploid success by promoting axillary branch elongation.

The maize doubled haploid (DH) technology plays an important role in accelerating breeding genetic gain. One major challenge in fully leveraging the potential of DH technology to accelerate genetic gain is obtaining a consistent seed return from haploid (DH0) plants after chromosome doubling. Here we demonstrated that DH0 seed production can be increased by increasing the number of mature axillary female inflorescences (ears) at anthesis. To determine the maximum capacity of a maize plant to develop ears, we first characterized the developmental progression of every axillary meristem. We found that all axillary meristems developed to a similar developmental stage before the reproductive transition of the shoot apical meristem (SAM). Upon reproductive transition of the SAM, all axillary meristems are released for reproductive development into ears in a developmental gradient reflective on their positions along the main stem. However, under most circumstances only the top one or two ears can generate silks at anthesis. We found that applying the GA inhibitor paclobutrazol (PAC) during the early reproductive transition of axillary meristems increased the number of silking ears at anthesis, leading to increased success of self-pollination and seed production. These results provide a blueprint to improve DH efficiency and demonstrate the potential of breeding innovation through understanding crops' developmental processes.

Control of meristem determinacy by trehalose 6-phosphate phosphatases is uncoupled from enzymatic activity.

Meristem fate is regulated by trehalose 6-phosphate phosphatases (TPPs), but their mechanism of action remains mysterious. Loss of the maize TPPs RAMOSA3 and TPP4 leads to reduced meristem determinacy and more inflorescence branching. However, analysis of an allelic series revealed no correlation between enzymatic activity and branching, and a catalytically inactive version of RA3 complements the ra3 mutant. Together with their nuclear localization, these findings suggest a moonlighting function for TPPs.

Signaling from maize organ primordia via FASCIATED EAR3 regulates stem cell proliferation and yield traits.

Shoot apical meristems are stem cell niches that balance proliferation with the incorporation of daughter cells into organ primordia. This balance is maintained by CLAVATA-WUSCHEL feedback signaling between the stem cells at the tip of the meristem and the underlying organizing center. Signals that provide feedback from organ primordia to control the stem cell niche in plants have also been hypothesized, but their identities are unknown. Here we report FASCIATED EAR3 (FEA3), a leucine-rich-repeat receptor that functions in stem cell control and responds to a CLAVATA3/ESR-related (CLE) peptide expressed in organ primordia. We modeled our results to propose a regulatory system that transmits signals from differentiating cells in organ primordia back to the stem cell niche and that appears to function broadly in the plant kingdom. Furthermore, we demonstrate an application of this new signaling feedback, by showing that weak alleles of fea3 enhance hybrid maize yield traits.

The Maize PI/GLO Ortholog Zmm16/sterile tassel silky ear1 Interacts with the Zygomorphy and Sex Determination Pathways in Flower Development.

In monocots and eudicots, B class function specifies second and third whorl floral organ identity as described in the classic ABCE model. Grass B class APETALA3/DEFICIENS orthologs have been functionally characterized; here, we describe the positional cloning and characterization of a maize (Zea mays) PISTILLATA/GLOBOSA ortholog Zea mays mads16 (Zmm16)/sterile tassel silky ear1 (sts1). We show that, similar to many eudicots, all the maize B class proteins bind DNA as obligate heterodimers and positively regulate their own expression. However, sts1 mutants have novel phenotypes that provide insight into two derived aspects of maize flower development: carpel abortion and floral asymmetry. Specifically, we show that carpel abortion acts downstream of organ identity and requires the growth-promoting factor grassy tillers1 and that the maize B class genes are expressed asymmetrically, likely in response to zygomorphy of grass floral primordia. Further investigation reveals that floral phyllotactic patterning is also zygomorphic, suggesting significant mechanistic differences with the well-characterized models of floral polarity. These unexpected results show that despite extensive study of B class gene functions in diverse flowering plants, novel insights can be gained from careful investigation of homeotic mutants outside the core eudicot model species.

Regulatory modules controlling maize inflorescence architecture.

Genetic control of branching is a primary determinant of yield, regulating seed number and harvesting ability, yet little is known about the molecular networks that shape grain-bearing inflorescences of cereal crops. Here, we used the maize (Zea mays) inflorescence to investigate gene networks that modulate determinacy, specifically the decision to allow branch growth. We characterized developmental transitions by associating spatiotemporal expression profiles with morphological changes resulting from genetic perturbations that disrupt steps in a pathway controlling branching. Developmental dynamics of genes targeted in vivo by the transcription factor RAMOSA1, a key regulator of determinacy, revealed potential mechanisms for repressing branches in distinct stem cell populations, including interactions with KNOTTED1, a master regulator of stem cell maintenance. Our results uncover discrete developmental modules that function in determining grass-specific morphology and provide a basis for targeted crop improvement and translation to other cereal crops with comparable inflorescence architectures.

The maize Gα gene COMPACT PLANT2 functions in CLAVATA signalling to control shoot meristem size.

Shoot growth depends on meristems, pools of stem cells that are maintained by a negative feedback loop between the CLAVATA pathway and the WUSCHEL homeobox gene. CLAVATA signalling involves a secreted peptide, CLAVATA3 (CLV3), and its perception by cell surface leucine-rich repeat (LRR) receptors, including the CLV1 receptor kinase and a LRR receptor-like protein, CLV2 (ref. 4). However, the signalling mechanisms downstream of these receptors are poorly understood, especially for LRR receptor-like proteins, which lack a signalling domain. Here we show that maize COMPACT PLANT2 (CT2) encodes the predicted α-subunit (Gα) of a heterotrimeric GTP binding protein. Maize ct2 phenotypes resemble Arabidopsis thaliana clavata mutants, and genetic, biochemical and functional assays indicate that CT2/Gα transmits a stem-cell-restrictive signal from a CLAVATA LRR receptor, suggesting a new function for Gα signalling in plants. Heterotrimeric GTP-binding proteins are membrane-associated molecular switches that are commonly activated by ligand binding to an associated seven-pass transmembrane (7TM) G-protein-coupled receptor (GPCR). Recent studies have questioned the idea that plant heterotrimeric G proteins interact with canonical GPCRs, and our findings suggest that single pass transmembrane receptors act as GPCRs in plants, challenging the dogma that GPCRs are exclusively 7TM proteins.

Chaperonins facilitate KNOTTED1 cell-to-cell trafficking and stem cell function.

Cell-to-cell communication in plants includes the selective trafficking of transcription factors and other signals through plasmodesmata. The KNOTTED1 (KN1) homeobox (KNOX) family transcription factors, which use this pathway, are essential for stem cell establishment and/or maintenance. Here we show that KN1 trafficking requires the chaperonin complex, which belongs to a group of cytosolic chaperones that fold specific substrate proteins. Genetic and physical interaction data show a functional relevance for chaperonins in KNOX family-dependent stem cell maintenance. Furthermore, tissue-specific complementation assays indicate a mechanistic basis for chaperonin function during the posttranslocational refolding process. Our study shows that chaperonins are essential for the cell-to-cell trafficking of a subset of mobile transcription factors and demonstrates the importance of chaperonin-dependent protein trafficking for plant stem cell function.

Digital gene expression signatures for maize development.

Genome-wide expression signatures detect specific perturbations in developmental programs and contribute to functional resolution of key regulatory networks. In maize (Zea mays) inflorescences, mutations in the RAMOSA (RA) genes affect the determinacy of axillary meristems and thus alter branching patterns, an important agronomic trait. In this work, we developed and tested a framework for analysis of tag-based, digital gene expression profiles using Illumina's high-throughput sequencing technology and the newly assembled B73 maize reference genome. We also used a mutation in the RA3 gene to identify putative expression signatures specific to stem cell fate in axillary meristem determinacy. The RA3 gene encodes a trehalose-6-phosphate phosphatase and may act at the interface between developmental and metabolic processes. Deep sequencing of digital gene expression libraries, representing three biological replicate ear samples from wild-type and ra3 plants, generated 27 million 20- to 21-nucleotide reads with frequencies spanning 4 orders of magnitude. Unique sequence tags were anchored to 3'-ends of individual transcripts by DpnII and NlaIII digests, which were multiplexed during sequencing. We mapped 86% of nonredundant signature tags to the maize genome, which associated with 37,117 gene models and unannotated regions of expression. In total, 66% of genes were detected by at least nine reads in immature maize ears. We used comparative genomics to leverage existing information from Arabidopsis (Arabidopsis thaliana) and rice (Oryza sativa) in functional analyses of differentially expressed maize genes. Results from this study provide a basis for the analysis of short-read expression data in maize and resolved specific expression signatures that will help define mechanisms of action for the RA3 gene.

Differentiating Arabidopsis shoots from leaves by combined YABBY activities.

In seed plants, leaves are born on radial shoots, but unlike shoots, they are determinate dorsiventral organs made of flat lamina. YABBY genes are found only in seed plants and in all cases studied are expressed primarily in lateral organs and in a polar manner. Despite their simple expression, Arabidopsis thaliana plants lacking all YABBY gene activities have a wide range of morphological defects in all lateral organs as well as the shoot apical meristem (SAM). Here, we show that leaves lacking all YABBY activities are initiated as dorsiventral appendages but fail to properly activate lamina programs. In particular, the activation of most CINCINNATA-class TCP genes does not commence, SAM-specific programs are reactivated, and a marginal leaf domain is not established. Altered distribution of auxin signaling and the auxin efflux carrier PIN1, highly reduced venation, initiation of multiple cotyledons, and gradual loss of the SAM accompany these defects. We suggest that YABBY functions were recruited to mold modified shoot systems into flat plant appendages by translating organ polarity into lamina-specific programs that include marginal auxin flow and activation of a maturation schedule directing determinate growth.

Stage-specific regulation of Solanum lycopersicum leaf maturation by class 1 KNOTTED1-LIKE HOMEOBOX proteins.

Class 1 KNOTTED1-LIKE HOMEOBOX (KNOXI) genes encode transcription factors that are expressed in the shoot apical meristem (SAM) and are essential for SAM maintenance. In some species with compound leaves, including tomato (Solanum lycopersicum), KNOXI genes are also expressed during leaf development and affect leaf morphology. To dissect the role of KNOXI proteins in leaf patterning, we expressed in tomato leaves a fusion of the tomato KNOXI gene Tkn2 with a sequence encoding a repressor domain, expected to repress common targets of tomato KNOXI proteins. This resulted in the formation of small, narrow, and simple leaves due to accelerated differentiation. Overexpression of the wild-type form of Tkn1 or Tkn2 in young leaves also resulted in narrow and simple leaves, but in this case, leaf development was blocked at the initiation stage. Expression of Tkn1 or Tkn2 during a series of spatial and temporal windows in leaf development identified leaf initiation and primary morphogenesis as specific developmental contexts at which the tomato leaf is responsive to KNOXI activity. Arabidopsis thaliana leaves responded to overexpression of Arabidopsis or tomato KNOXI genes during the morphogenetic stage but were largely insensitive to their overexpression during leaf initiation. These results imply that KNOXI proteins act at specific stages within the compound-leaf development program to delay maturation and enable leaflet formation, rather than set the compound leaf route.

The flowering hormone florigen functions as a general systemic regulator of growth and termination.

The florigen paradigm implies a universal flowering-inducing hormone that is common to all flowering plants. Recent work identified FT orthologues as originators of florigen and their polypeptides as the likely systemic agent. However, the developmental processes targeted by florigen remained unknown. Here we identify local balances between SINGLE FLOWER TRUSS (SFT), the tomato precursor of florigen, and SELF-PRUNING (SP), a potent SFT-dependent SFT inhibitor as prime targets of mobile florigen. The graft-transmissible impacts of florigen on organ-specific traits in perennial tomato show that in addition to import by shoot apical meristems, florigen is imported by organs in which SFT is already expressed. By modulating local SFT/SP balances, florigen confers differential flowering responses of primary and secondary apical meristems, regulates the reiterative growth and termination cycles typical of perennial plants, accelerates leaf maturation, and influences the complexity of compound leaves, the growth of stems and the formation of abscission zones. Florigen is thus established as a plant protein functioning as a general growth hormone. Developmental interactions and a phylogenetic analysis suggest that the SFT/SP regulatory hierarchy is a recent evolutionary innovation unique to flowering plants.

The NGATHA distal organ development genes are essential for style specification in Arabidopsis.

Floral organ identities are specified by a few transcription factors that act as master regulators. Subsequently, specification of organ axes programs the distribution of distinct tissue types within the organs that themselves develop unique identities. The C-class, AGAMOUS-clade MADS box genes are primary promoters of the gynoecium, which is divided into a distal style and a subtending ovary along the apical-basal axis. We show that members of a clade of B3 domain transcription factors, NGATHA1 (NGA1) to NGA4, are expressed distally in all lateral organs, and all four have a redundant and essential role in style development. Loss of all four genes results in gynoecia where style is replaced by valve-like projections and a reduction in style-specific SHATTERPROOF1 (SHP1) expression. In agreement, floral misexpression of NGA1 promotes ectopic style and SHP1 expression. STYLISH1, an auxin biosynthesis inducer, conditionally activated NGA genes, which in turn promoted distal expression of other STY genes in a putative positive feedback loop. Inhibited auxin transport or lack of YABBY1 gene activities resulted in a basally expanded style domain and broader expression of NGA genes. We speculate that early gynoecium factors delimit NGA gene response to an auxin-based signal, elicited by STY gene activity, to restrict the activation of style program to a late and distal carpel domain.

A protracted and dynamic maturation schedule underlies Arabidopsis leaf development.

Leaf development has been monitored chiefly by following anatomical markers. Analysis of transcriptome dynamics during leaf maturation revealed multiple expression patterns that rise or fall with age or that display age-specific peaks. These were used to formulate a digital differentiation index (DDI) based on a set of selected markers with informative expression during leaf ontogeny. The leaf-based DDI reliably predicted the developmental state of leaf samples from diverse sources and was independent of mitotic cell division transcripts or propensity of specific cell types. When calibrated by informative root markers, the same algorithm accurately diagnosed dissected root samples. We used the DDI to characterize plants with reduced activities of multiple CINCINNATA (CIN)-TCP (TEOSINTE BRANCHED1, CYCLOIDEA, PCF) growth regulators. These plants had giant curled leaves made up of small cells with abnormal shape, low DDI scores, and low expression of mitosis markers, depicting the primary role of CIN-TCPs as promoters of differentiation. Delayed activity of several CIN-TCPs resulted in abnormally large but flat leaves with regular cells. The application of DDI has therefore portrayed the CIN-TCPs as heterochronic regulators that permit the development of a flexible and robust leaf form through an ordered and protracted maturation schedule.

Signals derived from YABBY gene activities in organ primordia regulate growth and partitioning of Arabidopsis shoot apical meristems.

Shoot apical meristems (SAMs) are self-sustaining groups of cells responsible for the ordered initiation of all aerial plant tissues, including stems and lateral organs. The precise coordination of these processes argues for crosstalk between the different SAM domains. The products of YABBY (YAB) genes are limited to the organ primordium domains, which are situated at the periphery of all SAMs and which are separated by a margin of three to seven cells from the central meristem zone marked by WUSCHEL and CLAVATA3 expression. Mutations in the two related YAB1 genes, FILAMENTOUS FLOWER and YABBY3 (YAB3), cause an array of defects, including aberrant phyllotaxis. We show that peripheral YAB1 activity nonautonomously and sequentially affects the phyllotaxis and growth of subsequent primordia and coordinates the expression of SAM central zone markers. These effects support a role for YAB1 genes in short-range signaling. However, no evidence was found that YAB1 gene products are themselves mobile. A screen for suppression of a floral YAB1 overexpression phenotype revealed that the YAB1-born signals are mediated in part by the activity of LATERAL SUPPRESSOR. This GRAS protein is expressed at the boundary of organ primordia and the SAM central zone, distinct from the YAB1 expression domain. Together, these results suggest that YAB1 activity stimulates signals from the organs to the meristem via a secondary message or signal cascade, a process essential for organized growth of the SAM.

The tomato FT ortholog triggers systemic signals that regulate growth and flowering and substitute for diverse environmental stimuli.

The systemic model for floral induction, dubbed florigen, was conceived in photoperiod-sensitive plants but implies, in its ultimate form, a graft-transmissible signal that, although activated by different stimuli in different flowering systems, is common to all plants. We show that SFT (SINGLE-FLOWER TRUSS), the tomato ortholog of FLOWERING LOCUS T (FT), induces flowering in day-neutral tomato and tobacco plants and is encoded by SFT. sft tomato mutant plants are late-flowering, with altered architecture and flower morphology. SFT-dependent graft-transmissible signals complement all developmental defects in sft plants and substitute for long-day stimuli in Arabidopsis, short-day stimuli in Maryland Mammoth tobacco, and light-dose requirements in tomato uniflora mutant plants. The absence of donor SFT RNA from flowering receptor shoots and the localization of the protein in leaf nuclei implicate florigen-like messages in tomato as a downstream pathway triggered by cell-autonomous SFT RNA transcripts. Flowering in tomato is synonymous with termination of the shoot apical meristems, and systemic SFT messages attenuate the growth of apical meristems before and independent of floral production. Floral enhancement by systemic SFT signals is therefore one pleiotropic effect of FT orthologs.

Endogenous and synthetic microRNAs stimulate simultaneous, efficient, and localized regulation of multiple targets in diverse species.

Recent studies demonstrated that pattern formation in plants involves regulation of transcription factor families by microRNAs (miRNAs). To explore the potency, autonomy, target range, and functional conservation of miRNA genes, a systematic comparison between plants ectopically expressing pre-miRNAs and plants with corresponding multiple mutant combinations of target genes was performed. We show that regulated expression of several Arabidopsis thaliana pre-miRNA genes induced a range of phenotypic alterations, the most extreme ones being a phenocopy of combined loss of their predicted target genes. This result indicates quantitative regulation by miRNA as a potential source for diversity in developmental outcomes. Remarkably, custom-made, synthetic miRNAs vectored by endogenous pre-miRNA backbones also produced phenocopies of multiple mutant combinations of genes that are not naturally regulated by miRNA. Arabidopsis-based endogenous and synthetic pre-miRNAs were also processed effectively in tomato (Solanum lycopersicum) and tobacco (Nicotiana tabacum). Synthetic miR-ARF targeting Auxin Response Factors 2, 3, and 4 induced dramatic transformations of abaxial tissues into adaxial ones in all three species, which could not cross graft joints. Likewise, organ-specific expression of miR165b that coregulates the PHABULOSA-like adaxial identity genes induced localized abaxial transformations. Thus, miRNAs provide a flexible, quantitative, and autonomous platform that can be employed for regulated expression of multiple related genes in diverse species.