Transient efflux inhibition improves plant regeneration by natural auxins.

Plant genome editing and propagation are important tools in crop breeding and production. Both rely heavily on the development of efficient in vitro plant regeneration systems. Two prominent regeneration systems that are widely employed in crop production are somatic embryogenesis (SE) and de novo shoot regeneration. In many of the protocols for SE or shoot regeneration, explants are treated with the synthetic auxin analog 2,4-dichlorophenoxyacetic acid (2,4-D), since natural auxins, such as indole-3-acetic acid (IAA) or 4-chloroindole-3-acetic acid (4-Cl-IAA), are less effective or even fail to induce regeneration. Based on previous reports that 2,4-D, compared to endogenous auxins, is not effectively exported from plant cells, we investigated whether efflux inhibition of endogenous auxins could convert these auxins into efficient inducers of SE in Arabidopsis immature zygotic embryos (IZEs). We show that natural auxins and synthetic analogs thereof become efficient inducers of SE when their efflux is transiently inhibited by co-application of the auxin transport inhibitor naphthylphthalamic acid (NPA). Moreover, IZEs of auxin efflux mutants pin2 or abcb1 abcb19 show enhanced SE efficiency when treated with IAA or efflux-inhibited IAA, confirming that auxin efflux reduces the efficiency of Arabidopsis SE. Importantly, in contrast to the 2,4-D system, where only 50-60% of the embryos converted to seedlings, all SEs induced by transport-inhibited natural auxins converted to seedlings. Efflux-inhibited IAA, like 2,4-D, also efficiently induced SE from carrot suspension cells, whereas IAA alone could not, and efflux-inhibited 4-Cl-IAA significantly improved de novo shoot regeneration in Brassica napus. Our data provides new insights into the action of 2,4-D as an efficient inducer of plant regeneration but also shows that replacing this synthetic auxin for efflux-inhibited natural auxin significantly improves different types of plant regeneration, leading to a more synchronized and homogenous development of the regenerated plants.

The central role of stem cells in determining plant longevity variation.

Vascular plants display a huge variety of longevity patterns, from a few weeks for several annual species up to thousands of years for some perennial species. Understanding how longevity variation is structured has long been considered a fundamental aspect of the life sciences in view of evolution, species distribution, and adaptation to diverse environments. Unlike animals, whose organs are typically formed during embryogenesis, vascular plants manage to extend their life by continuously producing new tissues and organs in apical and lateral directions via proliferation of stem cells located within specialized tissues called meristems. Stem cells are the main source of plant longevity. Variation in plant longevity is highly dependent on the activity and fate identity of stem cells. Multiple developmental factors determine how stem cells contribute to variation in plant longevity. In this review, we provide an overview of the genetic mechanisms, hormonal signaling, and environmental factors involved in controlling plant longevity through long-term maintenance of stem cell fate identity.

Endogenous auxin maintains embryonic cell identity and promotes somatic embryo development in Arabidopsis.

Somatic embryogenesis (SE), or embryo development from in vitro cultured vegetative explants, can be induced in Arabidopsis by the synthetic auxin 2,4-dichlorophenoxyacetic acid (2,4-D) or by overexpression of specific transcription factors, such as AT-HOOK MOTIF NUCLEAR LOCALIZED 15 (AHL15). Here, we explored the role of endogenous auxin [indole-3-acetic acid (IAA)] during 2,4-D and AHL15-induced SE. Using the pWOX2:NLS-YFP reporter, we identified three distinct developmental stages for 2,4-D and AHL15-induced SE in Arabidopsis, with these being (i) acquisition of embryo identity; (ii) formation of pro-embryos; and (iii) somatic embryo patterning and development. The acquisition of embryo identity coincided with enhanced expression of the indole-3-pyruvic acid auxin biosynthesis YUCCA genes, resulting in an enhanced pDR5:GFP-reported auxin response in the embryo-forming tissues. Chemical inhibition of the indole-3-pyruvic acid pathway did not affect the acquisition of embryo identity, but significantly reduced or completely inhibited the formation of pro-embryos. Co-application of IAA with auxin biosynthesis inhibitors in the AHL15-induced SE system rescued differentiated somatic embryo formation, confirming that increased IAA levels are important during the last two stages of SE. Our analyses also showed that polar auxin transport, with AUXIN/LIKE-AUX influx and PIN-FORMED1 efflux carriers as important drivers, is required for the transition of embryonic cells to proembryos and, later, for correct cell fate specification and differentiation. Taken together, our results indicate that endogenous IAA biosynthesis and its polar transport are not required for the acquisition of embryo identity, but rather to maintain embryonic cell identity and for the formation of multicellular proembryos and their development into histodifferentiated embryos.

miR156-independent repression of the ageing pathway by longevity-promoting AHL proteins in Arabidopsis.

Plants age by developmental phase changes. In Arabidopsis, the juvenile to adult vegetative phase change (VPC) is marked by clear heteroblastic changes in leaves. VPC and the subsequent vegetative to reproductive phase change are promoted by SQUAMOSA PROMOTOR BINDING PROTEIN-LIKE (SPL) transcription factors and repressed by miR156/157 targeting SPL transcripts. By genetic, phenotypic, and gene expression analyses, we studied the role of the longevity-promoting AT-HOOK MOTIF NUCLEAR LOCALIZED 15 (AHL15) and family members in SPL-driven plant ageing. Arabidopsis ahl loss-of-function mutants showed accelerated VPC and flowering, whereas AHL15 overexpression delayed these phase changes. Expression analysis and tissue-specific AHL15 overexpression revealed that AHL15 affects VPC and flowering time directly through its expression in the shoot apical meristem and young leaves, and that AHL15 represses SPL2/9/13/15 gene expression in a miR156/157-independent manner. The juvenile traits of spl loss-of-function mutants appeared to depend on enhanced expression of the AHL15 gene, whereas SPL activity prevented vegetative growth from axillary meristem by repressing AHL15 expression. Our results place AHL15 and close family members together with SPLs in a reciprocal regulatory feedback loop that modulates VPC, flowering time, and axillary meristem development in response to both internal and external signals.

The end of flowering: interactions between cytokinin and regulatory genes.

Although the molecular regulation of global proliferative arrest (GPA) in arabidopsis (Arabidopsis thaliana) has been studied extensively, the precise role of the different contributors and their interconnections requires further research. A recent contribution by Merelo et al. now provides evidence that repression of cytokinin (CK) signaling affects the promotion of GPA.

Control of cambium initiation and activity in Arabidopsis by the transcriptional regulator AHL15.

Plant secondary growth, which is the basis of wood formation, includes the production of secondary xylem, which is derived from meristematic cambium cells embedded in vascular tissue. Here, we identified an important role for the Arabidopsis thaliana (Arabidopsis) AT-HOOK MOTIF CONTAINING NUCLEAR LOCALIZED 15 (AHL15) transcriptional regulator in controlling vascular cambium activity. The limited secondary xylem development in inflorescence stems of herbaceous Arabidopsis plants was significantly reduced in ahl15 loss-of-function mutants, whereas constitutive or vascular meristem-specific AHL15 overexpression produced woody inflorescence stems. AHL15 was required for enhanced secondary xylem formation in the woody suppressor of overexpression of constans 1 (soc1) fruitfull (ful) double loss-of-function mutant. Moreover, we found that AHL15 induces vascular cambium activity downstream of the repressing SOC1 and FUL transcription factors, most likely similar to how it enhances lateral branching by promoting biosynthesis of the hormone cytokinin. Our results uncover a novel pathway driving cambium development, in which AHL15 expression levels act in parallel to and are dependent on the well-established TDIF-PXY-WOX pathway to differentiate between herbaceous and woody stem growth.

An Arabidopsis AT-hook motif nuclear protein mediates somatic embryogenesis and coinciding genome duplication.

Plant somatic cells can be reprogrammed into totipotent embryonic cells that are able to form differentiated embryos in a process called somatic embryogenesis (SE), by hormone treatment or through overexpression of certain transcription factor genes, such as BABY BOOM (BBM). Here we show that overexpression of the AT-HOOK MOTIF CONTAINING NUCLEAR LOCALIZED 15 (AHL15) gene induces formation of somatic embryos on Arabidopsis thaliana seedlings in the absence of hormone treatment. During zygotic embryogenesis, AHL15 expression starts early in embryo development, and AH15 and other AHL genes are required for proper embryo patterning and development beyond the globular stage. Moreover, AHL15 and several of its homologs are upregulated and required for SE induction upon hormone treatment, and they are required for efficient BBM-induced SE as downstream targets of BBM. A significant number of plants derived from AHL15 overexpression-induced somatic embryos are polyploid. Polyploidisation occurs by endomitosis specifically during the initiation of SE, and is caused by strong heterochromatin decondensation induced by AHL15 overexpression.

Intervessel pit membrane thickness best explains variation in embolism resistance amongst stems of Arabidopsis thaliana accessions.

BACKGROUND AND AIMS: The ability to avoid drought-induced embolisms in the xylem is one of the essential traits for plants to survive periods of water shortage. Over the past three decades, hydraulic studies have been focusing on trees, which limits our ability to understand how herbs tolerate drought. Here we investigate the embolism resistance in inflorescence stems of four Arabidopsis thaliana accessions that differ in growth form and drought response. We assess functional traits underlying the variation in embolism resistance amongst the accessions studied using detailed anatomical observations. METHODS: Vulnerability to xylem embolism was evaluated via vulnerability curves using the centrifuge technique and linked with detailed anatomical observations in stems using light microscopy and transmission electron microscopy. KEY RESULTS: The data show significant differences in stem P50, varying 2-fold from -1.58 MPa in the Cape Verde Island accession to -3.07 MPa in the woody soc1 ful double mutant. Out of all the anatomical traits measured, intervessel pit membrane thickness (TPM) best explains the differences in P50, as well as P12 and P88. The association between embolism resistance and TPM can be functionally explained by the air-seeding hypothesis. There is no evidence that the correlation between increased woodiness and increased embolism resistance is directly related to functional aspects. However, we found that increased woodiness is strongly linked to other lignification characters, explaining why mechanical stem reinforcement is indirectly related to increased embolism resistance. CONCLUSIONS: The woodier or more lignified accessions are more resistant to embolism than the herbaceous accessions, confirming the link between increased stem lignification and increased embolism resistance, as also observed in other lineages. Intervessel pit membrane thickness and, to a lesser extent, theoretical vessel implosion resistance and vessel wall thickness are the missing functional links between stem lignification and embolism resistance.

A suppressor of axillary meristem maturation promotes longevity in flowering plants.

Post-embryonic development and longevity of flowering plants are, for a large part, determined by the activity and maturation state of stem cell niches formed in the axils of leaves, the so-called axillary meristems (AMs)1,2. The genes that are associated with AM maturation and underlie the differences between monocarpic (reproduce once and die) annual and the longer-lived polycarpic (reproduce more than once) perennial plants are still largely unknown. Here we identify a new role for the Arabidopsis AT-HOOK MOTIF NUCLEAR LOCALIZED 15 (AHL15) gene as a suppressor of AM maturation. Loss of AHL15 function accelerates AM maturation, whereas ectopic expression of AHL15 suppresses AM maturation and promotes longevity in monocarpic Arabidopsis and tobacco. Accordingly, in Arabidopsis grown under longevity-promoting short-day conditions, or in polycarpic Arabidopsis lyrata, expression of AHL15 is upregulated in AMs. Together, our results indicate that AHL15 and other AHL clade-A genes play an important role, directly downstream of flowering genes (SOC1, FUL) and upstream of the flowering-promoting hormone gibberellic acid, in suppressing AM maturation and extending the plant's lifespan.