Engineering Plant Cell Fates and Functions for Agriculture and Industry

Abstract

Many plant species are grown to enable access to specific organs or tissues, such as seeds, fruits, or stems. In some cases, a value is associated with a molecule that accumulates in a single type of cell. Domestication and subsequent breeding have often increased the yields of these target products by increasing the size, number, and quality of harvested organs and tissues but also via changes to overall plant growth architecture to suit large-scale cultivation. Many of the mutations that underlie these changes have been identified in key regulators of cellular identity and function. As key determinants of yield, these regulators are key targets for synthetic biology approaches to engineer new forms and functions. However, our understanding of many plant developmental programs and cell-type specific functions is still incomplete. In this Perspective, we discuss how advances in cellular genomics together with synthetic biology tools such as biosensors and DNA-recording devices are advancing our understanding of cell-specific programs and cell fates. We then discuss advances and emerging opportunities for cell-type-specific engineering to optimize plant morphology, responses to the environment, and the production of valuable compounds.

Figure 1

Many plants are cultivated for specific organs, tissues, or cells, which have been targets for selective breeding. A. Changes in inflorescence size and architecture and a compact plant morphology contributed to yield increases in both Oryza glaberrima (African rice) and Oryza sativa (Asian rice). B. The domestication of Gossypium hirsutum (cotton) increased the length and number of seed coat hairs as well as the ease of detachment. C. Many plant natural products are found in trichomes, including artemisinin found in the leaf glandular trichomes of Artemisia annua (sweet wormwood) (right), and tetrahydrocannabinolic acid (THCA) found in the stalked glandular trichomes of female Cannabis sativa L (cannabis) flowers (left). Increasing the trichome density has been used as a strategy to increase product yields.

Figure 2

Examples of biosensors deployed in plants A. FRET biosensors for hormone detection. In the absence of the hormone, excitation results in emission of the blue fluorophore. The presence of the hormone enables energy transfer between the two fluorophores, leading to the emission from the green fluorophore. B. HACR (Hormone Activated Cas9 Repressor) biosensors are composed of dCas9 fused to hormone-activated degron, and transcriptional repressor (topless; TPL) domains. In the absence of hormone, the HACR associates with its sgRNA, binding to and repressing the promoter of the output/reporter module. In the presence of the hormone, the HACR is degraded, leading to derepression. C. A riboswitch biosensor for theophylline. The RNA aptamer is encoded in the 3′ untranslated region of the output/reporter module (bottom). In the absence of theophylline, the aptamer conformation occludes the RNA cleavage site and results in translation (top-left). In the presence of theophylline (red), a conformational change exposes the cleavage site, leading to RNA-degradation (top-right).

Figure 3

DNA-recording devices create heritable memory, allowing cell lineages to be traced. A. A CRISPR-Cas9-based DNA-recording device composed of an out-of-frame fluorescent protein (FP) with a gRNA target site at the 5′ extremity of the coding sequence. The promoter controlling Cas9 expression is activated by molecular signals (transcription factors) in progenitor cells, leading to insertion/deletion events, some of which restore the reporter reading frame, enabling the detection of daughter cells by sequencing or FP visualization. B. An integrase-based DNA-recorder for analysis of cambium stem cells. Expression of an integrase (Cre) is activated in cambium stem cells in the presence of dexamethasone (DEX). The integrase mediates the excision of the terminator (T) in the output module, leading to the expression of the FP. C. Integrase-editable memory by engineered mutagenesis with optical in situ readout (intMEMOIR). Implemented in mammalian cell cultures, this method uses three-state memory elements (trits) in which no editing = no expression; inversion = expression of barcode 1; and excision = expression of barcode 2. Multiple occurrences of this unit are present in each cell, and their state is read using smFISH. After the induction of integrase expression, DNA editing will accumulate over time, and cell lineages can be recapitulated by analyzing the state of these multiple units. This enables the retracing of the cell-lineage trees.

Figure 4

Engineering plant development and performance. A. Auxin-repression of axillary bud development is dependent on the auxin-mediated activation of the auxin transporter PIN-FORMED1 (PIN1). A synthetic hormone-activated Cas9-based repressor (HACR) decreased the activation of expression of PIN1 by auxin, reducing feedback and leading to fewer side branches. B. Expression of a gain-of-function mutation in the developmental regulator INDOLE-3-ACETIC ACID INDUCIBLE 14 (IAA14) called solitary root (slr-1) eliminates root branching but also hinders root gravitropism, root hair development, and primary root growth (left). Lateral-root-stem-cell-specific expression (center) restores gravitropism, root hair development, and primary root growth. Expression of slr-1 (right) was tuned by controlling expression via cell-type-specific expression of a synthetic transcription factor (AmtR-VP16) and a cognate synthetic promoter with one, two, four, and six copies of the AmtR operator, enabling control over branching. C. Leaf stomata open in light and close in the dark to limit water-loss (left). To accelerate stomatal kinetics, a synthetic potassium channel fused to a blue-light-triggered photoreceptor (BLINK1) was expressed from a guard cell-specific promoter. This increased growth under fluctuating white light mimics the temporary shade conditions experienced by passing clouds or shadows.

Figure 5

Strategies for increasing natural-product yields via cell-type-specific engineering. A. Increased densities of glandular secretory trichomes in A. annua via network engineering to tune the abundance of developmental regulators AaMIXTA1 (MIXTA-Like 1), AaHD1 (HD-ZIP IV 1), and AaHD8 (HD-ZIP IV 8). B. The vinblastine biosynthetic pathway is divided into five subpathways, spatially separated across four cell types in Catharanthus roseus (left). The MEP pathway (1) and Iridoid pathway (2) are predominantly in phloem-associated parenchyma cells. The late iridoid pathway (3) and alkaloid pathway are in epidermal cells, and the late alkaloid pathway (5) in idioblast cells (brown) and laticifer cells. Yield increases (right, purple annotations) might be achieved via (i) developmental reprogramming to increase laticifer branching or idioblast density, (ii) upregulation of 1-deoxy-d-xylulose 5-phosphate synthase (DXS) in phloem-associated parenchyma, (iii) upregulation of the rate-limiting acetyl-CoA:4-O-deacetylvindoline 4-O-acetyltransferase (DAT) in idioblast and laticifer cells, and (iv) expression of horseradish peroxidase in idioblast or laticifers to aid oxidative coupling of catharanthine and vindoline.